MlCHlGAN STAYE UNlVERSlTY LIBRARIE 13% e d 5 0‘5 a L. lllllllll Iiliili’iill Ii lilflllllillnlliil w _ 3 1293 00606 1646 LIBRARY Michigan State University—J l I This is to certify that the thesis entitled Improving the Experimental Skills of High School Biology Students by Introducing Laboratory Techniques of Molecular Biology presented by Mary Margaret Fowler has been accepted towards fulfillment of the requirements for Master of Science degree in Biological Sciences Major professor weir 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE _—‘fi 4i {31—5—9422}; 1 MC ,i__l| l = —_l l l MSU Is An Affltmdive Action/Equal Opportunity Institution IMPROVING THE EXPERIMENTAL SKILLS OF HIGH SCHOOL BIOLOGY STUDENTS BY INTRODUCING LABORATORY TECHNIQUES OF MOLECULAR BIOLOGY by Mary Margaret Fowler A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Interdepartmental Biological Sciences 1989 to r n N ABSTRACT IMPROVING THE EXPERIMENTAL SKILLS OF HIGH SCHOOL BIOLOGY STUDENTS BY INTRODUCING LABORATORY TECHNIQUES OF MOLECULAR BIOLOGY by Mary Margaret Fowler The problem addressed by this thesis is: can the skills and techniques used in molecular biology and biotechnology be adapted for use in the high school curriculum? Two modules. or units. were developed. taught to high school students. and analyzed. These modules taught the concepts and laboratory techniques of spectrophotometry, protein electrophoresis, pipetting, pH, buffers. enzyme characterisitics, sterile technique. plant tissue culture, bacterial gene regulation. and bacterial transformation using a plasmid. The modules were introduced into the existing biology curriculum by means of lecture, laboratory exercises. supplementary readings, audio visuals, and discussion. Highly significant differences were found between the three pre-tests and corresponding post-tests. It was found to be clearly feasible to expect high school students to use and understand the concepts and techniques presented. The students demonstrated conclusively that they can handle the equipment and concepts. ACKNOWLEDGMENTS Many people have given me assistance and support in the course of my studies. I would like to thank and express appreciation to the following: The masters committee: Dr. Clarence Suelter for his encouragement and help in developing the direction of the program and thesis, and Dr. Howard Hagerman and Dr. Martin Hetherington for their support and for serving on the committee. My fellow students. who began the National Science Foundation Workshop in Molecular Biology in June 1987, who shared ideas, tested and revised laboratory exercises. and gave their support at critical times. My son Steven A., and daughter Karen L., for their considerable encouragement. My husband, Harold E. Fowler, without whose enduring emotional support and confidence this thesis would not have been possible. iv TABLE OF CONTENTS page LIST OF TABLES ............... . ..................... vii LIST OF FIGURES.. ..... .. .......................... viii Chapter 1. INTRODUflIONOOOO ...... 00.0.... 000000000000000000 1 General Description of the Modules ........ . ..... 1 Literature Survey. ...... ....................... 2 Central Question to be Answered by Modules ...... 5 Research 0bJectives........... ............. ...6 Real World Obiects Addressed by Modules.. ....... 7 Content ObJectives.. .......... ................ 8 Analysis of Literature used in Creating and Teaching the Modules........ ........ .11 II. TRANSFORAMTION.... ..... .. ...................... 14 Analysis of Clinical Interview ................. 14 Survey of Entire Group of Students ............. 20 Independent Research ProJect... ................ 24 III. INSTRUCTION.. .................................. 28 Daily Calendar ................... . ............. 28 Outline of Lessons ............................. 29 Techniques Used in the Laboratory .............. 31 Titles and ObJectives for Laboratory Exercises.37 IV. EVALUATION.... ......... . ....................... 40 Analysis of Data ............................... 40 V. CONCLUSIONS .................................... 49 Adaptation of Techniques to High School ........ 49 Maintain Emphasis on Inquiry Process Instruction....... ......... .... ............. 50 Application of Techniques to Independent Research ProJect ..... . ........... . .......... 51 Weaknesses and Strengths ...... . .......... . ..... 52 APPENDICES A. LABORATORY EXERCISES..................... ...... 53 B. TEACHER’S GUIDES TO LABORATORY EXERCISES.......74 C. DETAILED OUTLINE OF LESSONS ........... . ........ 85 n. PRE-TESTS AND POST-TESTS ...................... 124 E. SUPPLEMENTARY HANDOUTS ................ . ....... 14S BIBLIOGRAPHY............. ............ . ........... ..153 vi LIST OF TABLES Clinical Exit Interviews.......................15 Use of Lab Techniques in Independent Research PrOJectOOOOO'OOIOOOQOOOCOO000.000.000.0000017 Results of Student Surveys for second hour following Biotechnology Module.... ......... 21 Results of student surveys for fourth hour following Biotechnology Module..... ...... ..22 Module I Part 1 Pro-post Tests........ ......... 41 Module I Part 2 Pre-post Tests.......... ....... 42 Module II Biotechnology Pre-posts Tests.. ...... 43 Summary Results of Pro-Tests and Post-Tests....44 vii LIST OF FIGURES 1. Availability of Laboratory Exercises. ......... .53 viii CHAPTER 1 INTRODUCTION WES There is a need to develop scientifically and technologically literate individuals. Perhaps scientific literacy can be improved by incorporating advances in molecular biology and biotechnology into the high school curriculum. The question addressed by this thesis is: can the skills and techniques used in molecular biology be adapted for use in the high school curriculum? This thesis describes the development and teaching of two modules stressing laboratory techniques. The first module concentrates on introductory chemistry for beginning biological science students. This module of 26 days includes important concepts related to carbohydrates. lipids. proteins. and enzymes. Spectrophotometry, pipetting, electrophoresis and pH and buffers are introduced in this module. The second module. 16 days in length, concentrates on DNA and biotechnology. Sterile technique is introduced in this module and applied to plant tissue culture, lac operon gene regulation in Esgbgnighiaflggli and an introduction to recombinant DNA by the transformation of E £911 using a plasmid. These modules were taught to two sections of sophomore level honors biology students at Clarkston Senior High School in Clarkston, Michigan. These students were selected using the Otis-Lennon and Metropolitan Science Achievement Test scores. The course is intended for scientifically gifted and talented students. Although the purpose of these modules is to provide technological knowledge in the area of molecular biology, the emphasis on inquiry process skills still continues to be important. The final unit of the year, which is an independent research proJect requiring library research. a controlled scientific experiment and a formal research report, was retained. The independent research proJect represents an opportunity for students to apply the concepts, process skills and lab techniques that they have learned during the year. W The role of the science teacher is changing. Many feel the primary role of teachers is preparing future citizens to live more effectively in a scientific technological society. Citizens should be able to use scientific or technological information to make the important decisions society demands (Yager, 1986). Some also feel that science teachers should encourage students to become the next generation of scientists. Bassam Shakhashiri. National Science Foundation director for science and engineering education. states “We have a dual mission, to attract students to careers in science and to make sure the general population is scientifically literate“ (McCormick 1989). The National Science Teachers Association issued a policy statement that addresses these needs. The following is taken from the NSTA Position Statement 12894:: “The aim of science education for the 1980’s is to develop scientific and technological literacy for all citizens. This aim emphasizes a general understanding of science and technology including knowledge, processes, applications, and information concerning opportunities for those interested in careers related to science and engineering. Science education for the 1980’s should achieve five fundamental goals: a) to develop scientific and technological process and inquiry skills: b) to provide scientific and technological knowledge: c) to use the skills and knowledge of science and technology as they apply to personal and social decisions: d) to enhance the development of attitudes, values, and appreciation of science and technology: and e) to study the interactions among science-technology-society in the context of science-related societal issues.“0_ 0030. 00: m 0:: m 00:00:00 0:: a_0>0_ 0.00.5 0:» 0pc v 0:0 0 00:0osu0 «nu:0osun _0>0_ L000: 0:» 0:: a 0:0 > 00:00:0m .> 0.0:9 00> 00> 00> 00> 00> 00> o 00> 00> 00> 00> 00> 00> h :_0uoca «0 0:00 :00:000_0 00:00:000_0 :_000: a: 00500000.0 m 00> 00> 00> 00> 00> 00> m o:_:0.0 :00000 0:. 00:00:000_0 0000 0.200.: :_ 00:0500: 0:0: v 0000.:5000 0.0000e0 :0 0:::0.0 bonus: m0 =0 as. ..e eye—:00:— m 00000.0 00000.0 00000.0 0000 0:0: 0:0: 00:0 :0 00000.: :00000 00_ N u:_:0_0 o.seo_0 0:_:0_0 00:00:000_0 :00000 00. o:.:0_0 u :0.»00=c m w v o N a _0>0_ 0030. _0>0_ 0.00.3 _0>0_ 000:: 0:0osum 3u~>muaz~ Huxu acuuznqo .« manta 16 In response to the first question. three of the six students said the lab on tissue culture or cloning ‘was their favorite labifrom.both modules. The top student felt tissue culture had a lot of applications to the real world and it was interesting to see that a whole organism can arise from a little piece of tissue or single organ. The other two students also felt that it was the most interesting lab activity. The second question asked if any of the lab techniques were used in the individual research proJect. Four of six interviewed students used at least one of the techniques. and all of the four used pipettes. When the entire class was surveyed. nineteen of the 46 or 418 of the class used pipettes and pi-pumps. the pipetting devices. in their research proJect. Initially the students found the pipettes and pi-pumps fun to use. A few students were not sure they were using the pipettes correctly. but most had no problem. In past years. students would have used small graduate cylinders to measure solutions. but with the pipettes and pi-pumps conveniently available in a labeled drawer. they were used. Only an occasional student would ask for assistance. Other techniques were represented among the six interviewed students. These techniques included spectrophotometry. sterile technique in lac operon. and pH. Refer to Table 2 for 17 the results of techniques used by the entire class. Pipettes. spectrophotometry and pH from Module I were used most often. It was most pleasing to note that 11 students or 24k used the spectrophotometer as part of the research proJects. The 11 students needed little or no help blanking in the instrument or understanding the difference between percent transmittance and absorbance. A few felt more comfortable using optical density in place of absorbance. however. all but one of the 11 interpreted and plotted the data with no difficulty. TABLE 2. USE 0? LAD TECHNIQUES IN INDEPENDENT RESEARCH PROJECT Technique Number of students Percent Pipettes/pi-pumps 19 41% pH and/or buffers 14 30% Spectrophotometer 11 24% Tissue culture 3 6.5% _— Electrophoresis 1 2% -—I Sterile technique for 1 2k lac operon y Trable 2. Presented here are the results of a survey taken following the independent research proJect at the end of the year to determine the application of new concepts and techniques. 18 Regarding the third question. pH was thought to be most valuable in the future. The reasons given were that pH is important in understanding the use of household materials and important for next year’s chemistry class. The top student felt inoculating bacteria was the most important technique. This student was very interested in gene regulation. and had read a recent Scientific American article on the subJect. She felt that sterile technique and gene regulation were important to the future of biology. One student thought cloning was most valuable because it had practical applications and one student felt all the techniques were important. There was no agreement among the students on the most difficult concept to understand. The top student. after long reflection. felt she understood all of the techniques and concepts. The other 5 students each had different problem areas and different reasons. One of the middle students admitted she did not understand how the spectrophotometer worked and therefore did not understand the lab related to this technique. This student did not use any new technique in the individual research proJect. One student had most difficulty with electrophoresis because he couldn’t see the relationship to material discussed before the lab. 19 All six students felt that they had a more realistic view of cloning and recombinant DNA than when they began in September. Many of these students have typically read material on the subJect and a number of them held some serious misconceptions about these processes. The maJority felt that they understood what these techniques are. how they work. and how practical they can be. One student admitted that cloning might frighten her more now that she can see how easy it is to do. One of the top students had read the Monsanto booklet 'Of the Earth: Agriculture and the New Biology“ three or four times. and was fascinated by the ability to make plants frost resistant and other such applications. She felt she had a more realistic view because she saw the techniques as something she could be working on in the future. In response to the sixth question. three of the students replied that electrophoresis was least valuable. The reason given by two of the students was that they understood the lab but they did not Understand how it connected to other material from the class. This is a deficiency that will need to be addressed in the future. All of the students felt that the process skills twill be valuable to them in the future. They felt they iaould need to use these in college classes. 20 When working with students of this ability. it is necessary to design the course to force students to use higher level thinking skills. With this in mind. the last question asked was if they were challenged to think. All six students responded with a definite yes. One of the top student’s response was “In any class I can drift through. I did have to think in here. But. I wanted to think about it.“ The other top student’s response was “When I walk into most of my classes. I feel like I’m memorizing. I didn’t feel like I was memorizing in here: I felt like I was putting together a puzzle." WIS At the end of the Module II. the following student . survey was given to all students. They were told not put their name on the survey and to write comments. STUDENT SURVEY 1. On a scale from 1 to 10 with 10 being excellent. how do you rate the Biotechnology unit? 2. On a scale of 1 to 10 with 10 being excellent. how much did you enJoy using the lab equipment? 3. Are you afraid of any of the organisms used in the lab. or do you have any doubts about accidently creating potentially dangerous “bugs“? 4. Do you plan a career in biology or another science? If so. what? 5. Which lab was your favorite? 21 .0.:: 0:0 :. 00. 00.0o>0m 0 00; 0:0 00:0.00 :. 000000 0 00::0.0 .050.:0o0o 0:» 000000 >05» 0. 00:00 00.0 0003 >059 .u:050.:oe 0:» mo 00: 0:0 0:0 .0.:: >mo.o:£oouo.0 000 0000 00 00x00 0003 00:00:00 000 .>0>0:0 0.00 c. .m 0.009 m:.:o.u o: o: o o «N 0.0000:000000.0 >50:00000 o: 0. m .N o:.:o.0 o: o: o. 0. ON o:.:o.o o: o: o. o. o. 00. p.500.0 0: o: b o o. m:.:o.0 o: o: m m 9. 0:0: 0: o: o. . b. it: o: o: 0. . m. :00000 00. o: o: w o v. o:.:o.o 0:.00:.u:0 00000O000 o: o o m. u:.:o.o o: o: 9 9 N. o:.:o.o o: o: o. o .. a:.:o.0 o: o: m w o. o:.:o.o o: o: o. o o o:.:o.o o: o: o 0 Q 0:.co.o o: o: o. m.o 9 :00000 00. 00>0E o: o. 9 m o:.:o.o 0030000.£000 .00.0>£0 o: m m m o:.:o.o 00>0E o: o v c o:.:o.0 o: o: o o m o:.:o.o >uo.oe0£u00 o: o o N o:.:o.0 >000.Eonuo.0 o: o 0.0 . m v m N a 000532 00005:: :o.000:o 0:00:00 0.000: >oo.o:£0000.0 o:.3o..o« 0:0: Ocooom 000 >0>030 0:00:00 mo 00.300m .0 0.009 22 .0.:: 000 :. 00. 00.0o>00 0 000 0:0 00:0.00 :. 000000 0 00::0.0 .050.:000o 000 000000 >000 0. 00000 00.0 0003 >009 .0:050.:00 000 00 00: 000 0:0 .0.:: >oo.o:00000.0 000 0000 00 00000 0003 00:00:00 000 .>0>0:0 0.00 :. .v 0.009 0:.:0.0 0: 0: 9 9 .N m:.:0.0 0: o: 0 0 ON it: 0: 00> m w o. 0:.:0.0 0: 0: o 0.0 0. :00000 00. 00000000 .00.005 0: 0. 0 9. o:.:o.0 0: o: 0 m 0. o:.:o.0 o: 0: N N m. 0:.:0.0 0: 0: 0. v v. o:.:o.0 >00.0000000 o: o. o. m. o:.:0.0 0:05000:05 >0000000 o: 9 m N. 0:.:0.0 :0.00>000000 00..0..3 00> 9 m 0. o:.:o.0 >ao.o.0 0:.005 0: 0 0 o. o:.:o.0 00.00:0o o: 9 9 0 m:.:o.0 >50:00000 0: 9 9 0 iii: >00000 .00:05:00.>:0 o: m.0 o 9 o:.:o.0 00.0>0o .o:.000:.o:0 0: 0. 0 0 0.500.: :0.00:.0000> 0: 0. 0 w ill: 0: 0: 9 o v m:.:o.0 >00.00:0:u0:.0.005 0: o 9 m m:.:o.0 0: 0: 00 9 N 0.00000000000.0 o: o: o. 0 0 m v m N . 0005:: 00005:: :o.000:c 0:00:00 0.:002 >00.o:00000.n o:.30..00 0:0: 000200 000 >0>0=0 0:00:00 00 00.0002 .0 0.005 23 Although Module II on Biotechnology was the most difficult unit of the course. the students gave the unit a passing grade of 7. They seemed to have have enJoyed using the equipment more than they valued the entire unit. but the overall response was positive (see Tables 3 and 4). Only two students were afraid of the organisms according to the survey and yet no one expressed a concern about safety during lab. Adapting the laboratory exercises for 55 minute periods was a challenge and caused students to feel rushed. Some of the comments were: more lab. more worksheets. hard to understand at first. its perfect. more electrophoresis. more field trips. more time for student questioning. its awesome. and more plasmid information. A few of the students commented that their parents were impressed by the unit. Since these students were identified as scientifically gifted. a question was included on career plans. In second hour. 5 of the 22 students or 23 3 completing the survey indicated an interest in a science career. This number was 11 out of 21 or 52 k for forth hour. Combining the two classes. this represents 35 a of the students have stated interest in science careers . 24 W The final unit of the year is an independent research proJect requiring library research. a controlled experiment and a formal research report. Approximately 5 weeks is devoted to this proJect. The first step. and often the most difficult for the student. is selection of a topic. The student is asked to formulate one or more questions that will serve as the research problem. The problem should take the following format: I'What is (are) the effect(s) of (an independent variable) on (a measurable dependent variable) in (organism or part(s) ?' Examples of independent variables. dependent variables. and suitable organisms are discussed in class. A large variety of reference articles is made available in the classroom including many past articles from Amenlgan Biglggy_Igaghgn. The second step involves library research leading to a written report called a literature search. The literature search will be used in the introduction section of the formal research report and should lead the student to formulate a hypothesis. The student then designs an experimental test of the hypothesis. Quantification is expected wherever possible and it is to be presented in data tables and graphs. 25 Considerable time is spent throughout the year on the process skills of formulating and using hypotheses. designing experiments. making data tables. constructing and interpreting graphs. controlling variables and drawing inferences. It is expected that the research proJect reflect these skills. The article “Writing the Scientific Paper in the Investigative Lab“ Amenlgan Biglggy_1eaghgn January 1977 is used as a guide to writing the formal research paper. A few weeks before work begins on the research proJect. the class is lead through the writing of a-practice report. The experiment that is used is “What is the effect of gibberellic acid on the germination rate of barley seeds?" Two reference articles are provided dealing with the role of gibberellic acid in stimulating protein production in the aluerone layer of barley seeds. These articles are thoroughly discussed in class. Information in these articles should be used to make inferences about the observed data. A maJor emphasis for both the gibberellic acid report and the final research report is the drawing of inferences. Inferring is not only a basic skill in science. but also a process that is fundamental to interpreting events in our everyday lives. Students are taught that an inference is an explanation of observations. In drawing an inference. the writer uses 26 any knowledge related to the subJect to produce the best explanation of an observation. Inferences are logical conclusions reached by reasoning. however. inferences are tentative and may not be true. In the final research report. the information gained by the literature search in the library is used by the student to draw the best possible inference at that time. Some of this year’s research proJects are: 1' What is the effect of a change in the concentration of copper ions on Chlorella? What is the effect of acid rain on growth of tomato plants? How can electrophoresis be used to separate plant proteins? What is effect of indoleacetic acid on the growth of decapitated tomato plants? What is the effect of radiation on germination of marigold seeds? How does increased amounts of carbon dioxide affect the temperature of the earth’s atmosphere? What are effects of gibberellic acid on dwarf and normal pea plants? What is the effect of iron deficiency on chlorosis of sunflower seedlings? 10. 11. 12. 13. 14. 15. 27 What is the effect of pH on the growth rate of Chlorella? What are buffering effects of soil limestone? What is the effect of acid rain on earthworms? What is the effect of 2.4-D on monocots versus dicots? What is the effect of increased carbon dioxide on the growth rate of chlorella? What are the effects of growth regulators on the culture of cauliflower tissue? What is effect of lactose concentration on gene regulation in Lac Operon? Refer to Table 2 for a summary of the use of the laboratory techniques in the independent research proJect. Seventeen of 23 or 74% of second hour used at least one of the techniques. Fourteen of 23 or 61% of fourth hour applied one technique. This represents 67% for both classes combined. CHAPTER 3 INSTRUCTION WEAR Wu The first module was taught in 26 teaching days. Preceding this module. 6 days were spent on introducing inquiry process skills of formulating and using hypotheses. designing experiments. controlling variables. making data tables. constructing and interpreting graphs. The test given on day 9 of the module was the students’ first test of the school year and included questions related to the process skills. A unit on cells and cell function followed the module. Day 1 Pre-test. Introductory Chemistry Day 2 Chemical Model Building Day 3 pH - Acids. Bases. Indicators Day 4 Laboratory exercise: Discovery of pH Using Common Household Substances Day 5 Laboratory exercise: Effect of Buffers on the pH of Solutions Day 6 Acid Rain Day 7 Video Tape “Acid Rain" Day 8 Discussion Day 9 Test 1 (Post test) Part 2 Organic Chemistry Day 10 Pre-test. Carbohydrates Day 11 Dehydration synthesis and hydrolysis of carbohydrates Day 12 Lipids Day 13 Proteins Day 14 Proteins. Enzymes Day 15 Laboratory exercise: Testing for Organic Molecules Day 16 Laboratory exercise. continued 28 29 Day 17 Laboratory exercise: Efffect of pH Protein Digestion by Papain Day 18 Introduction to Spectrophotometry and Pipettes Day 19 Laboratory exercise: Effect of temperature activity of salivary amylase Day 20 Laboratory exercise: Determining Concentration of Protein in Milk Day 21 Laboratory exercise: continued Day 22 Introduction to Electrophoresis Day 23 Laboratory exercise: Protein Electrophoresis Day 24 Laboratory exercise. continued Day 25 Discussion Day 26 Test (Post test) The second module required 16 days. Preceding this module was an extensive unit on DNA stucture and function and heredity. A unit on bioethics followed the module. Day 1 Cloning Day 2 Preparation for lab Day 3 Laboratory exercise: Effects of Two Plant Hormones on Culture of African Violet Tissue Day 4 Gene Regulation Day 5 Laboratory exercise: Effect of Lactose on Lac Operon: A Study in Gene Regulation Day 6 Complete Laboratory exercise and discussion Day 7 Recombinant DNA Day 8 VCR “Life Patent Pending“ Day 9 Preparation for lab Day 10 Laboratory exercise: Transformation of the Bacteria Egghenighia gall with pBR322 Plasmid Day 11 Laboratory exercise. continued Day 12 VCR Geometry of Life Day 13 VCR Monsanto Day 14 VCR edited segments with discussion Day 15 Review Day 16 Test W A brief outline of both modules is presented here. More detail with references to visuals and other supporting materials is presented in the Appendix C. 30 MODULE I I. Introductory chemistry A. Chemical elements of life B. Structure of Atoms C. Chemical bonds 1. covalent bond 2. ionic bond D. Molecules E. Water F. Acids and bases 1. Dissociation and the pH scale 2. pH indicators G. Buffers H. Homeostasis II. Biological chemistry A. Inorganic and organic molecules B. Structure of Organic molecules C. Carbohydrates 1. Monosaccharides 2. Disaccharides 3. Polysaccharides 4. Dehydration synthesis and hydrolysis 5. Functions D. Lipids 1. Fatty acids 2. Dehydration synthesis and hydrolysis 3. Functions E. Proteins 1. Amino acids 2. Protein structure 3. Dehydration synthesis and hydrolysis 4. Functions F. Enzymes 1. Enzymes-substrate complexes 2. Mechanism of action 3. Factors that affect enzyme activity MODULE II I. Gene regulation in bacteria A. Inducible systems 1. RNA polymerase 2. Promoter 3. Structural genes 4. Regulator gene 5. Operator B. Lac Operon II. Biotechnology A. Cloning 1. Totipotency 31 2. Differentiation 3. Callus 4. Applications B. Recombinant DNA 1. Restriction enzymes 2. Ligase enzymes 3. Plasmids 4. Applications 5. Concerns W The following is a list of laboratory techniques being used in the instruction of the two modules described earlier: 1. Spectrophotometry a) standard curve 2. Electrophoresis 3. Pipetting 4. Concept of pH and buffers 5. Sterile technique applied to: a) plant tissue culture b) bacterial gene regulation c) bacterial transformation A spectrophotometer is an instrument which can measure the transmittance of light through a substance or. can measure the absorbance of light by a substance. The denser a substance is the more light it will absorb and the less it will transmit. Spectrophotometry is a useful tool for biochemists because many compounds of biochemical interest either are colored or react with a 32 substance to give it a color. The colored product will absorb light of a particular wavelength which can be measured in a spectrophotometer. In the first lab using spectrophotometry. the amount of light tramsitted through samples of starch and iodine was measured. The amount of light transmitted varies directly with the concentration of the starch in the solution. As the enzyme hydrolyzes (digests) the starch. there is less starch in the test tube to absorb the light. therefore more light will be transmitted. The technique is used to examine the affects of temperature on the activity of amylase. In the second lab using spectrophotometry. the protein concentration in milk is determined. Determining protein concentrations in biological tissues is an important component of many molecular biology experiments. The concentration of pure proteins is often determined by reacting it with a substance to give it a color. Proteins will react with Biuret solution to produce a purple color. The concentration of the purple product formed by the interaction of the protein and Biuret solution can be measured in a spectrophotometer at 562 nanometers. A standard curve is made using known concentrations of protein. By plotting the known concentrations of protein against the absorbance. a straight line should i 33 be obtained. As the concentration increases. the absorbance increases. From this standard curve the concentration of an unknown protein in solution can be determined . Electrophoresis is the movement of charged molecules in an electric field. Electrophoresis separates by taking advantage of the fact that different molecules have different electrical charges which causes them to travel in different directions or' in the same directions but at different distances in an electrical field. When the substances are placed in a separating medium like agarose and submerged in a solution that conducts electricity the molecules will move toward one of the poles. Electrophoresis is often used to separate charged molecules such as proteins and DNA. Protein electrophoresis can be used to detect sickle cell anemia and to determine if a heart attack has occured. Electrophoresis of DNA can be used in conJunction with standard genetic means to map chromosomes and in law enforcement. to match body fluid samples with those of suspects or to.identify victims. Pipetting is useful in transferring small quantities of liquid from container to container. In today’s microtechniques fractions of a milliliter to ten or twenty milliliters are transferred. while in some cases. 10 - 20 milliliters may be handled at a 34 time. The amount depends upon the characteristics of the experiment. The liquid is carefully drawn into the pipette by creating a vacuum with a pipetting device such as a pi-pump. The pipette is used to transfer measured amount of liquid to the receiving container ‘and the contents allowed to run out. Another useful concept involves pH and buffers. An acid is a substance that releases hydrogen ions when mixed with water. A base is a substance that releases hydroxide ions when mixed with water. Chemicals called pH indicators. such as bromthymol blue. can be used to determine the approximate pH. Organisms and cells can generally withstand variations in the pH of the external environment. In contrast. the internal environment of cells is very sensitive to pH change and take place in a medium in which the pH is carefully regulated. The control of an almost constant pH in living systems is achieved by buffers. A buffer is a substance capable of neutralizing both acids and bases in a solution and maintaining the original acidity or basicity of the solution. Buffers tend to keep the pH constant by taking up or releasing H+ ions or OH‘ as these are added to the solution. Buffers can resist pH changes due to metabolic production of acids such as lactic acid and bases such as ammonia. The maJor 35 buffering systems found in cellular fluids involve phosphate. bicarbonate. amino acids and proteins. Sterile technique allows one to transfer and grow bacteria or any other organism without contamination. Media. containers. pipettes and any other equipment used to handle the organisms must be sterilized before use. Knowing good sterile technique allows pure cultures of bacteria to grown for many applications. Tissue culture also requires sterile technique. Tissue culture. the growing of tissue on sterile nutrient media in the laboratory. has greatly facilitated research in both plant and animal developmental biology. The original plant and all its offspring form a clone. that is. they are all . genetically alike. A clone is a population of cells or individuals descended from one original cell or individual by asexual propagation. and therefore genetically indentical. When plant cells are grown in culture. they initially form a tumor-like mass called a callus. but when certain growth regulators are present in the media. the cells differentiate to form normal plant tissues. It is possible. therefore. under certain conditions. to grow complete plants from previously undifferentiated tissue. Three french scientists. Francois Jacob. Jacques Monod and Jean-Pierre Changeaux received the Nobel 36 Prize in Medicine in the late sixties for their work in helping to understand the way gene expression is regulated. They used the colon bacterium. E; ggli for which they were able to propose a mechanism for the action of a set of genes that are essential for lactose utilization. They termed the gene set the Lao Operon because it involved utilization of the sugar. lactose and an operator region on the DNA that served as a binding site for a regulatory molecule. It has subsequently been found that the operon consists of one or more structural genes capable of coding for specific enzymes. the operator. and the promoter which is a region of DNA next to the operator where RNA polymerase binds prior to transcription. A regulator gene that codes for a repressor protein is located near the operon and it too has a promoter associated with it. Recombinant DNA technology has greatly changed modern biology. One application of recombinant DNA in the laboratory is the transformation of ampicillin resistant bacteria to ampicillin sensitive. Ampicillin is a penicillin derivative that kills bacterial cells by interfering with cell wall synthesis. The ampicillin-resistance gene codes for an enzyme (beta-lactamase) that cuts and inactivates the antibiotic. Most E;,_ggli cells found in nature are ampicillin-sensitive and the effcts of the antibiotic 37 are therefore toxic. A widely used plasmid. pBR322. contains an ampicillin-resistance gene. that is. a gene for B-lactamase. that when introduced into ampicillin-sensitive cells. will transform them into ampicillin resistant bacteria. W MODULE I PART 1 INTRODUCTORY CHEMISTRY 1. Discovery of pH Using Common Household Substances To demonstrate that indicators can be used to determine pH of a substance. To learn that different items have a different pH. 2. Effect of Buffers on the pH of a Solution To relate pH to acids. bases and salts. To relate the concept of pH to the buffering action and the maintainence of a selected pH. To express buffer action in terms of homeostasis in living systems. To graph data. MODULE I PART 2 ORGANIC CHEMISTRY 3. Testing for Organic Compounds To perform and interpret the result’s of the Benedict’s for reducing sugar. To perform and interpret the result’s of iodine test for starch. To perform and interpret the result’s of the Sudan 111 test and Brown Paper test for lipids. 38 To perform and interpret the result’s of the Biuret test for proteins. To use the specific chemical tests to determine the presence of sugar. starch. lipid. and protein in UNKNOWN FOOD SAMPLES. Effect of pH the Protein Digestion by Papain To follow the enzymatic breakdown of a protein. To determine the effect of pH on enzymatic action. Effect of Temperature on the Activity of Salivary Amylase To discover some of the physical changes that occur during starch digestion. To become familiar with the use of the Spec 20 or other colorimeter. To measure the effects of temperature on the speed of starch digestion. Determining Concentration of Protein in Milk Using Biuret To determine the concentration of proteins in milk. To gain experience using a pipette. To gain an appreciation of the use and operation of the spectrophotometer. To make and interpret a graph. To gain experience using a standard curve of known protein concentrations to interpolate the concentration of an unknown protein. Protein Electrophoresis To separate protein molecules by charge using electrophoresis in agarose gel. 10. 39 Effects of Two Plant Hormones on Culture of African Violet Leaf Tissue To recognize that different hormones affect plant cells in different ways to produce various types of growth. To practice sterile technique. To develop a practice of keeping accurate. consistent record keeping over long periods of time. Effect of lactose on the lac operon: A Study in Gene Regulation To determine if lactose will induce the production of B-galactosidase (lactase) in E. coli. To introduce students to sterile technique. Transformation of the bacteria EgngL1,with the pBR322 Plasmid To transform the bacterium E.,gng from ampicillin sensitive to ampicillin resistant with the plasmid pBR322. CHAPTER 4 EVALUATION W Two groups of tenth grade biology students identified as gifted and talented participated in the study. The classes are referred to as second hour and fourth hour. Each class began with 24 students enrolled in September. One student from second hour moved. and one from fourth hour dropped the class. leaving 23 students in each class. Therefore. the total sample size was 46. The first module was presented in two parts. with a pre-test and post-test administered for each part. while the second module had a single pre-test and post-test. The tests are included in Appendix D. In the first module part 1, the pre-test and post-test were exactly the same. The test reflected five of the ten stated content obJectives. These obJectives were related to structure of the atom. description of ions and ionic bonding. and explanation of pH. ObJectives taught in the module but not represented on the test included valence. covalent bonding. polar properties of water. buffers and homeostasis. These topics were tested on a longer unit test but were not tested on the eight item pre-post test. 40 41 Table 5. Module I Part 1 Pre-post Tests Second Hour Fourth Hour Student Pre-test Post-test Pre-test Post-test 1 4 8 4 8 2 4 8 5 8 3 2 7 6 7 4 4 8 2 6 5 2 8 5 8 6 6 8 5 7 7 4 7 2 8 8 5 8 2 8 9 4 8 4 8 10 4 7 3 6 11 3 8 1 8 12 3 6 1 8 13 1 6 5 8 14 2 6 4 8 15 3 6 4 8 16 6 8 4 8 17 2 8 3 5 18 4 7 1 5 19 2 7 4 7 20 4 8 3 7 21 3 8 2 8 22 3 4 3 7 23 1 6 2 7 Mean 3.30 7.17 3.26 7.3 t . 11,0500 t a 11.22%» 009 < 0.001 42 Table 6. Module I Part 2 Pre-post Tests Second Hour Fourth Hour Student Pre-test Post-test Pre-test Post-test 000mmaww» ..a N mmamaqmmmaammmmmqqqmqaw mmmmmmmmmqmmmmmmmmmmmus \l mmmmmamwmsmmwammmmmmmmm A QOQflmflQflmmmmmmflflflmmmflm Mean 5.17 7.52 . . 7. t a 5.050% t 8 9.41** A 3 ** p < 0.001 43 Table 7. Module II Biotechnology Pre-post Tests Second Hour Fourth Hour Student Pre-test Post-test Pre-test Post-test 1 4 7 3 6 2 1 5 5 8 3 2 8 5 7 4 2 7 3 8 5 3 5 1 6 6 2 7 0 6 7 2 5 5 7 8 4 8 3 6 9 2 8 0 7 10 3 5 4 7 11 3 6 3 7 12 4 8 2 7 13 3 8 O 4 14 3 6 7 8 15 4 7 0 7 16 5 6 2 8 17 3 5 3 6 18 2 6 2 7 19 0 7 5 8 20 4 8 5 7 21 4 7 6 6 22 4 8 3 3 23 0 5 2 4 mean 3.0 6.52 2.78 6.61 t a 7.11** t - 10.64** xx p < 0.001 414 Tobie 8. SUMMARY RESULTS OF FEE-TESTS AND POST-TESTS Pro-test Post-test almost .. .. Test possible x 3.11. x 3.11 t score Intro 01e- 2nd hour a 3.3 1.33 7.17 1.05 11.05" Intro Chen 4th hour 8 3.26 1.42 7.3 0.95 11.22" (roan Chen 2nd hour 8 5.17 1.43 7.52 0.93 5.05" argon mo- 4th hour a 1.7 1.22 7.13 0.71 9.41" Blotech 2nd hour a 3.0 1.90 6.52 1.31 7.11" Biotech 411. hour a 2.78 1.28 6.61 1.16 10.64" Ii p < 0.001 45 The results for the first part of Module I are shown in Table 5. The null hypothesis is there is no difference between the pre and post tests. The null hypothesis was reJected at the 0.001 level of probability. Statistically significant differences were found in the performance on the pre-test and post-tests. Students performed well on the longer unit test also, with a combined mean of 89.2%. In Module I Part 2. Organic Chemistry, 5 of the 14 content obJectives were tested on the pre-test. These obJectives are related the definition of organic molecules, their functions in cells, valences of consistuent atoms. structural formulas of organic molecules. The post-test covered the same obJectives with these exceptions. The post-test did not test for valences of atoms in organic molecules, and tested for two laboratory obJectives. These obJectives were to learn the use of a Spectrophotometer, and to learn to separate protein molecules by charge using electrophoresis. Again the null hypothesis was reJected at the 0.001 level of probability (see Table 6). When looking at the two items on the post-test that tested for laboratory obJectives, the following information is noted. Eighty percent of the 46 46 students answered question seven correctly. They knew that when using the spectrophotometer to measure the concentration of starch iodine mixture. that the percent transmittance increases as the concentration of starch decreases. Eighty three percent knew that the electrophoresis experiment separated proteins by charge of the proteins. 0n the longer unit test that followed the post-test, there were 40 obJective questions and five short answer questions. The mean on the test, for both classes combined. was 888. One of the short answer questions was “Explain how the specctrophotometer and the standard curve could be used to determine the concentration of protein in milk". Most students answered this question in detail. A sample student answer follows: “The Spectrophotometer and standard curve can be used to determine the concentration of protein in milk by taking a known protein and putting Biuret solution in it (different known amounts in different test tubes) and getting the transmitttance, then put Biuret solution in the milk and get the transmittance. then find the absorbance and graph the absorbance by the amount of protein in each tube and draw a straight line through the most points possible and this is the standard curve.‘ Extrapolate or interpolate the line to the absorbance of the milk and 47 draw a line down to the protein amounts and that is the concentration of protein in the milk.“ This response was typical of the answers to this question. The class discussion and laboratory reports would also indicate an understanding of the concept. In Module II. 5 of the 9 content obJectives were tested on the pre-test. These obJectives were related to RNA transcription and translation, cell differentiation. cloning using tissue culture, and use of plasmids. restriction enzymes and ligase enzymes in recombinant DNA experiements. The post-test covered the concept of totipotency and lactose induction of the synthesis of B-galactosidase in the lac operon in addition to the obJectives tested in the pre-test. Again the null hypothesis was reJected at the 0.001 level of probability (see Table 7). On the longer unit test, there were 39 obJective questions and four essays. The complete test is included in Appendix D. The mean on the test was 83.3%. One of the essay questions is: Assume that you are working for a large pharmaceutical company in their research division. Your goal is to produce insulin in large quantity by genetic engineering techniques. You are provided with a DNA fragment. containing the insulin gene, which had been isolated using restriction enzyme A. You know that the insulin gene is flanked on 48 both ends by recognition sites for B. another restriction enzyme. An E. coli plasmid with single B recognition site is available. What would you do? Describe all the steps of your experiemnt. One student’s reply to this question follows. “Recut the plasmid so that it has two 8 recognition sites. This will create sticky ends. and the DNA for insulin will be accepted more readily. Next you take a ligase enzyme and splice the DNA together permanently. Now you shock the E‘_§gli into accepting the new plasmid. Now you put it into a dish and let it grow until there is enough to place in a larger container.“ The students enJoyed this unit. They found the basic steps in recombinant DNA technology relatively easy to understand. CHAPTER 5 CONCLUSIONS W It is clearly feasible to expect high school students to use and understand the techniques of spectrophotometry. electrophoresis. pipetting. pH and buffers, tissue culture. gene regulation, and bacterial transformation. The students have demonstrated conclusively that they can handle the equipment. the concepts and the organisms without undue difficulty. Spectrophotometry showed students that instruments can be used to monitor reactions and growth in living organisms. Certain concepts. such as gene regulation. are much more difficult to understand. Most of the students understood gene regulation and felt that the study of DNA had much more significance. Similarily, the study of bacterial transformation made the study of recombinant DNA and its applications more relevant. The students no longer looked at recombinant DNA as something to fear. but rather as a technique that may bring solutions to problems. Tissue culture was the most popular lab. Ii was not only interesting to see new roots, stems and leaves appear. but tissue culture also helped the students accept that a whole organism could be grown from a piece of an organ that had been engineered by recombinant DNA. 49 50 W A very important part of the course is the application of the inquiry process skills. These skills include formulating and using hypotheses. controlling variables. designing experiments. making data tables. constructing and interpreting graphs. and drawing inferences. All of the these skills except inference writing are usually introduced and tested in the first unit of the year. then used and reinforced throughout the year. Module I Part 1 did not interfere with this practice. If anything. the addition of the two labs in Module I Part 1 strengthened these skills by providing real data to be plotted in the buffer lab. In Module I Part 2. the students were able to absorb the content. the new techniques and the inquiry process skills. The new techniques were pipetting. spectrophotometry. standard curves. and protein electrophoresis. The two spectrophotometry labs in particular allowed students to hypothesize, control variables, construct data tables and graphs. The student were able to understand standard curves and even interpolate without excessive difficulty. Therefore. in both parts of Module I. the effect of the lab techniques on inquiry process instruction was positive. 51 However. in Module II, Biotechnology, the lab techniques and equipment were considerably more difficult. Students were able to handle the new techniques but attention to process skills such as hypothesizing and drawing inferences was more difficult. They became concerned with operating the new equipment and simply recording the data. Naturally. with the increasing difficulty in lab techniques. more class time for discussion following the labs was necessary. -A possible explanation of this observation may be that inquiry-oriented laboratories often impose an overload on the short term memory of students who at the same time need to attend to new subJect matter concepts, and unfamiliar equipment (Linn, 1977). The value of introducing the techniques of Biotechnology far exceeds this short coming. When the time came for independent research proJects. the students were told that they could use any of the equipment that had been used that year. There was no pressure to use any equipment. Sixty seven percent of the students used at least one of the new techniques or equipment. 52 W Considering that this was a pilot year for the incorporation of these concepts and techniques. there were few problems. One of the weaknesses was that the students sometimes flet rushed to complete the laboratory exercises in a 55 minute class period. There were occasional problems with equipment blowing fuses. However. the students were generally patient and forgiving and the lessons went well. There will be some refining and additions to the units. For example, more articles from science Journals will be used with the students at appropriate times. The most obvious strength of the modules was the enthusiasm generated among the students. These concepts and techniques can definitely be managed by high school students and the opportunity should be provided. APPENDICES APPENDIX A LABORATORY EXERCISES 53 APPENDIX A LABORATORY EXERCISES FIGURE 1. Availability of Laboratory Exercises Laboratory Exercise Title Availability* 1. Discovery of pii Using Comon Household Substances 2. Effect of Buffers on the pH of a Solution 3. Testing for Organic Compounds 4. Effect of pH on the Protein Digestion by Papain 5. Effect of Temperature on the Activity of Salivary Amylase 6. Determining Concentration of Protein in Milk Using Biuret Solution 7. Protein Electrophoresis 8. Effects of Two Plant Hormones on Culture of African Violet Leaf Tissue 9. Effect of lactose on the lac operon: A Study in Gene Regulation 10. Transformation of the bacteria E._,§QL1,wlth the pBR322 Plasmid Disk Appendix A Appendix A Disk Disk Disk (modified) Disk Appendix A Appendix A Appendix A i Disk refers to B.R.A.I.N.S. Disk which was developed as a result of National Science Foundation Workshop at Michigan State University in the summers of 1987 and 1988. The disk is available from Dr. Clarence Suelter, Dept. of Biochemistry. Michigan State University. East Lansing, MI 48824. S4 EFFECT OF BUFFERS ON THE pH OF A SOLUTION INTRODUCTION: An acid is a substance that releases hydrogen ions when mixed with water. A base is a substance that releases hydroxide ions when mixed with water. Salt and water form when acids and bases react. This process is called neutralization. Organisms and cells can generally withstand variations in the pH of the external environment. In contrast. the internal environment of cells is very sensitive to pH change and take place in a medium the pH of which is carefully regulated. The maJority of processes in cells occur near neutral pH 7. The hydrolysis enzymes of lysosomes. however. have their maximum activity at a pH in the region of 5, the common pH following the death of a cell. The control of an almost constant pH in living systems is achieved by buffers. A buffer is a substance capable of neutralizing both acids and bases in a solution and maintaining the original acidity or basicity of the solution. Buffers tend to keep the pH constant by taking up or releasing 3+ ions or on— as these are added to the solution. Buffers can resist pH changes due to metabolic production of acids such as lactic acid and bases such as ammonia. The maJor 55 buffering systems found in cellular fluids involve phosphate. bicarbonate. amino acids and proteins. W3: To relate pH to acids. bases and salts. To relate the concept of pH to the buffering action and the maintenance of a selected pH. To express buffer action in terms of homeostasis in living systems. To graph data. MATERIALS: beakers pH 7 buffer distilled water .1 M NaOH dropping pipettes .1 M HCl pH paper sensitive to .5 or pH meter W: Handle HCL and NaOH with caution. If these reagents are spilled on skin rinse for 3 to 5 minutes in running water. PROCEDURE: Part 1 Effect of HCL and NaOH on pH of water. 1. Pour 25 mL of distilled water into small beaker. Record the initial pH of the distilled water. 2. Add one drop of .1M HCl. swirl to mix and record observed pH. 3. Continue adding at the one drop rate. stir and record observed pH. 4. Repeat this process using a second beaker of distilled water and .1M NaOH. 5. Graph data. 56 Part 2 Effect of HCl and NaOH of pH of buffer. 1. Pour 25 mL of buffer into small beaker. Record the initial pH. 2. Add one drop of .1M MCI. swirl and record pH. 3. Continue adding at the one drop rate. swirl and record observed pH. 4. Repeat this process using a second beaker of buffer and .1M NaOH. 5. Graph data. Distilled water I Buffer Drop I HCI I NaOH 1 Drop I HCl I NaOH 1 I J l I 11 I I l l 1 I _1 1 10 I I l I I l 57 W: What happened to the pH of the distilled water when you added HCl? 2. What happened to the pH of the distilled water when you added NaOH? 3. What effect did the buffer have when you added BC] or NaOH? 4. By definition. homeostasis is a steady state producing a constant internal environment. Read the section on homeostais in your text book. Discuss how this idea could be related to the buffering action of the blood or the cytoplasm of the cell. 5. In certain parts of the world. the soil contains limestone which can have a buffering affect for acid rain. Explain. 6. What are the consequences for soil which do not contain limestone? W: 1. Design an experiment using hemoglobin as a buffer. 2. Design an experiment the effect of HCl and NaOH on buffered aspirin and non-buffered aspirin. 3. Design an experiment testing the effect of limestone on soil pH. 58 TESTING FOR ORGANIC MOLECULES INIRQDUCILQN: Specific molecules are necessary for life processes such as growth. development. reproduction. maintenance. and repair. Scientists have developed a series of tests to determine the presence or absence of the building blocks of living things in food samples. In testing the substances and food stuffs available. you should realize that the materials had a biological origin--they were made by plants and animals. There are many molecules present in living organisms. However. you will perform tests for only four of these molecules: sugar, starch, fat and protein. 0313911233: To perform and interpret the result’s of the Benedict’s for reducing sugar. To perform and interpret the result’s of iodine test for starch. To perform and interpret the result’s of the Sudan III test and Brown Paper test for lipids. To perform and interpret the result’s of the Biuret test for proteins. To use the specific chemical tests to determine the presence of sugar. starch. lipid. and protein in UNKNOWN FOOD SAMPLES. 59 MATERIALS: test tubes test tube rack glucose solution Benedict’s solution starch solution iodine solution cooking oil pieces of brown paper Sudan III Bile salt solution egg albumin biuret reagent onion Juice potato Juice plain gelatin lemon gelatin apple several food samples, soaked and softened in water ME: 1. TEST FOR SUGARS: Put 40 drops of glucose solution in test tube. Add equal amount of Benedict’s solution. Heat SLOWLY over the flame of an alcohol lamp. keeping contents of tube in motion. Observe color. Use the symbol - if ther is no color change. Use + for green. ++ for yellow. +++ for orange. and ++++ for dull red. 2. Certain sugars. called reducing sugars. can be detected by the use of Benedict’s test. Glucose. fructose. maltose and lactose are examples of reducing sugars. SUCROSE is not a reducing sugar and will therefore not react with Benedict’s solution. 3. TEST FOR STARCH: Put 40 drops of starch in solution in a test tube. Add 4 or 5 drops of iodine solution. to the test tube. Note color changes. Starch (amylose) can be identified by its reaction with iodine solution. A deep blue-black color is added to starch. Use the symbol - if there is no starch. Use +, ++, +++ to indicate the intensity of the blue color if there is starch. 4. TEST FOR LIPIDS: One test for the presence of fat is to ub the sample on brown paper. Rub cooking oil over small area of paper. On another section of the paper spead a drop of water. Allow the paper to dry. Compare spots. The presence of fat is indicated by a tracslucent spot on the paper. Hold the paper toward a light to observe spot. 5. ANOTHER TEST FOR FAT is to add Sudan III to sample. Place 20 drops of cooking oil in a test tube. Add 20 drops of tap water. Add 1 drop of Sudan III. Shake the test tube gently. The presence of fat is indicated by a pink color throughout the cooking oil. 6O 6. TEST FOR PROTEINS is to add sodium hydroxide and copper sulfate solutions (biuret reagent) to a food sample. Place 40 drops of egg white (albumen) in a test tube. Add 10 drops of biuret reagent. The presence of protein is indicated by a change to purple color. CAUTION: Sodium hydroxide is corrosive: it will burn skin. Be careful when pouring Biuret into test tube. 7. If you bring any foods from home. make sure it is thoroughly macerated and soaked before testing. Clean the test tubes between tests. DATA TABLE 1. Tests for Chemical Molecules Molecule Chemical Test Description sugar starch lipid protein TABLE 2. Tests for Organic Molecules in Food Samples (+ for positive. - for negative) carbohydrates food tested sugar I starch lipids protein 61 W 1. Describe the color changes observed for a positive reaction with each of the following: Benedict’s solution. iodine solution. Sudan, and Biuret. 2. Why is it possible to get a negative test for sugar in a candy bar? 3. Discuss the difference in carbohydrate storage substance of onion and potato. 62 EFFECTS of TWO PLANT HORMONES on the CULTURE of AFRICAN VIOLET TISSUE INIRQDQQILQN Tissue culture, the growing of tissue on sterile nutrient media in the laboratory. has greatly facilitated research in both plant and animal that is. they are all genetically alike. A clone is a population of cells or individuals descended from one original cell or individual by asexual propagation. and therefore genetically indentical. When plant cells are grown in culture, they initially form a tumor-like mass called a callus. but when certain growth regulators are present in the media. the cells differentiate to form normal plant tissues. It is possible. therefore. under certain conditions. to grow complete plants from previously undifferentiated tissue. Indole acetic acid (auxin) and cytokinin are but two several plant growth regulators. Although each is needed in growth regulation of the plant.their comparative ratios greatly affect the direction of growth of the developing plant. If the two hormones are in balence, i.e. near equal quantities. the culture will form undifferentiated callus: while a large auxin to cytokinin ratio leads to excessive root growth. If the auxin to cytokinin ratio is low, the shoot will form more prevalently. In either case the actual 63 concentrations of the two hormones are very low at 1 mg/l or less. W: To determine comparative concentrations of auxin and cytokinin on growth and development of African violet tissue. W One set for every two people Disinfecting solution (Clorox/water, 1:10) with one drop Joy Sterile distilled rinse water (500 ml) Sterile 250 ml beaker 70% ethanol in squirt bottle Scapel with metal handle and #10 blade 2 pair forceps (1 pair 10 inches or longer) 1 sterile petri plate Alcohol lamp 4-25x150mm culture tubes with Modified Murashige/Skoog medium (20 ml/culture tube) African violet plants PROCEDURE: Each group of four students will make four African Violets cultures. There are four different hormone concentrations. one concentration for each piece of leaf. 1. Wash hands with soap. clean under nails. 2. Spray both surfaces of the leaf with 70% ethanol. 3. Select a healthy appearing upper leaf and remove it. 4. Place the cut leaf in a sterile beaker and cover with Clorox and Joy solution for 10 minutes. 5. Pour off the Clorox and rinse with 3 separate changes of sterile distilled water. 6. Place the disinfected leaf in a sterile petri dish. 64 7. Pass the scalpel blade trough the flame and while one partner holds the petri dish lid at an angle over the petri dish base.. cut equal sized squares (1cm 2) from the leaf. Try not to include the large leaf veins in the cut sections. 8. Flame the opening of a tube in preparation for the culture transfer. 9. Using long forceps, transfer a leaf section of each of the 4 culture tubes. Make sure that the leaf sections are in complete contact with the medium. Flame the opening of the tube and cap it immedietly after the transfer. 10. Using a stick on label. place your name , treatment and date started on each tube. REFERENCES Galston. A., P. Davies and R. Satter. (1980). Ihg Life_pf_tb:_finaen_21ant . 3rd Ed. Prentice Hall, Inc. Englewood Cliffs. Hagerman. H. (1987). I'The Effects of Two Plant Hormones on the Culture of Tobacco Tissue“. High School Experiments from NSF Molecular Biology Workshop at MSU. Lundberg. D. (1987). Cloning African Violets. In: When 54(4). 37-39. Murashinge T. and F. Skoog. (1962). A revised medium for rapid growth and bloassays with tobacco tissue cultures. Physigli__213nt‘, 15:473-479. 65 DATA SHEET Observations should be recorded on a weekly basis and a drawing of the contents of each tube is to be made biweekly. The recorded data and drawings must corrospond with the assigned tube numbers. Use this legend in recording your data. c-calus laincrease in size r-roots d-decrease s-shoots nc-no change t-death f-fungus contamination Starting date Date .18 IAA .2 K .0005 IAA 1.0 K "H EFFECT OF LACTOSE ON THE LAC OPERON: A STUDY OF GENE REGULATION W: Three french scientists. Francois Jacob. Jacques Monod and Jean-Pierre Changeaux recieved the Nobel Prize in Medicine in the late sixties for their work in helping to understand the way gene expression is regulated. They used the colon bacterium, Eshgnighia call for which they were able to propose a mechanism for the action of a set of genes that are essential for lactose utilization. They termed the gene set the Lao Operon because it involved utilization of the sugar. lactose and an operator region on the DNA that served as a binding site for a regulatory molecule. It has subsequently been found that the operon consists of one or more structural genes capable of coding for specific enzymes. the operator, and the promoter which is a region of DNA next to the operator where RNA polymerase binds prior to transcription. A regulator gene that codes for a repressor protein is located near the operon and it too has a promoter associated with it. When E. coli cells are given only glucose for their energy and materials source. they produce enzymes that reflect that metabolic activity. If. on the other hand, these same cells are placed on a medium 67 containing only lactose. they soon begin to make enzymes that enable them to utilize lactose. Because lactose appears to have ’caused’ the cells to produce the proper enzymes. it is called an inducer. It induced the genes of the organism to synthesize one or more enzymes that make it possible to use the energy source. In this case. three enzymes are produced a new and they are: galactosidase. galactoside permease, and acetyl transferase. all of which are necessary for hydrolysis and utilization of lactose. This laboratory exercise is designed to test the hypothesis that lactose can serve as an inducer molecule. W8 To determine if lactose will induce the production of B-galactosidase (lactase) in E. coil. To introduce students to sterile technique. MATERIALS: E. coil culture Inoculating loop Glucose broth (0.05% sugar) in a test tube lactose broth (0.05% sugar) in a test tube O-nitrophenyl-beta-D-galactopyranoside (O-NPG) Eye droppers Marker Incubator set at 37°C CAUTION: Use special caution whenever you are working with bacteria. Use sterile technique as demonstrated. 68 ERQQEDQRE: Day 1 1. Using sterile technique . inoculate each test tube with E. coli from the culture. (Use sterile technique as demonstrated) 2. Label each tube with its sugar content and your group number. 3. Place each tube in a rack and place them in the incubator overnight. Day 2 4. To each of the 2 incubated tubes. add 10 drops of the O-NPG solution. 5. Record the results every ten minutes and complete the data table below. Table 1. Results obtained from adding 10 drape of O-NPG to each of 2 culture tubes. Legend: + a strongly yellow: - = weakly yellow: 0 I no color produced beyond background color. 10 min. 20 min. 30 min. Lactose (flucom: 1. Do background research to determine what happened. (hint: O-NPG is an indicator for activity of the enzyme B—galactosidase). Write a conclusion. EHKUER;HMEEIUHUHQES 1. Use a spectrophotometer (SPEC 20) to track the color change for 30 minutes. 69 TRANSFORMATION OF THE BACTERIA ESCHERICHIA CQLLWITH THE pBR322 PLASMID INTRODUCTION: AnLlDlQLLQ_R::L:Lin£: Ampicillin is a penicillin derivative that kills bacterial cells by interfering with cell wall synthesis. The ampicillin-resistance gene codes for an enzyme (beta-lactamase) that cuts and inactivates the antibiotic. Most E‘_ggll cells found in nature are ampicillin-sensitive and the effcts of the antibiotic are therefore toxic. A widely used plasmid. pBR322. contains an ampicillin-resistance gene. that is. a gene for B-lactamase. that when introduced into ampicillin-sensitive cells, will transform them into ampicillin resistant bacteria. Transformation Introducing pBR322 plasmid into E‘_ggii cells will be accomplished in the following manner. 1. Treat the bacterial cells with calcium chloride to allow the cells to become more permeable and increase their ability to take up the plasmid. Note: Calcium chloride treated cells are said to COMPETENT 2. Incubate competent cells with plasmid DNA. The cells will take in the plasmid. 70 3. Show that the cells have taken in the plasmid by growing cells on an antibiotic-containing agar medium. If the cells have the plasmid they will live on the antibiotic. 0122112! An E._ggli strain that is sensitive to ampicillin will be made competent by treatment with ice cold 09012. E. ggli cells will be mixed and incubated with the pBR322 plasmid (+ DNA cells). As a control. “- DNA“ cells will be treated identically to the “+ DNA“ cells except they will not recieve the plasmid. The cells will be heat shocked to further help uptake of the plasmid and then incubated in a normal growth medium to allow the cells to recover. After recovery, some of the cells will be spread onto LB+amp plates which will select for transformed cells. Some cells will be spread on LB plates which allow both transformed and untransformed cells to grow. After an overnight incubation. the plates will be examined. Note: This experiment includes several steps requiring sterile technique. Review the procedures for sterile technique before beginning the experiment. A star indicates those steps that require sterile technique. 71 DBJECILEE To transform the bacterium Escherichia £911 from being ampicillin-sensitive to being ampicillin-resistant with the plasmid pBR322. MATERIALS per group of four students EL_§QLL (strain mm294) 24 hour culture on LB plates C3012. sterile and ice cold 2 sterile culture tubes with caps inculating loop 3 sterile pipets (plastic) one container of ice LB (Luria broth) liquid culture medium 1 LB agar plate 1 LB-ampicillin agar plate alcohol lamp test tube rack marker Your group and other groups must share the following materials: Plasmid with the Ampicillin-Resistance gene (+DNA) (pBR322 Plasmid) water bath. 42 C 37 C incubator PROCEEDURE: 1. Using a marker. label one sterile test tube “+DNA“ and a second sterile test tube “-DNA". Also, put an identifying mark of some kind on the cap of each tube. Stand the tubes in a test tube rack. 2. CAUTION! Sterile-wrapped pipets and loops must be opened from the end that will be held when using the unwrapped instrument. Open a pipet from its bulbous end: Opne a loop from.its pointed end. Never allow the unwrapped circular loop or narrow end of the pipet to contact any nonsterile obJect. Care must be taken to insure the sterility of these instruments prior to use. Immediately discard any used pipet or loop into the chlorox solution. *3. With one sterile pipet. transfer 250 ul (0.25 ml) 169“¢Old CAClz into the -DNA tube and then a like volume into the +DNA tube. (Figure 1). Stand both tubes in ice. Note: Because the caps on the culture tubes are not easily lifted. it is advisable that one *4. x6. *9. 10. 72 member of the your group handle the tubes. while another member does the pipetting. Obtain an LB plate with a 24 hour E‘_ggii culture. Select ten (10) isolated bacterial colonies from your plate and transfer them to the +DNA CaClz tube. This is quicker and easier to do if the 10 colonies are in a row so that you can pick up the cells with a single swipe across the plate. To make the resuspension of the cells easier. deposit the cells onto the side of the tube Just above the liquid. Replace the cap, then swirl gently to . Repeat with the -DNA tube. Place tubes in ice. Obtain the vial of plasmid DNA. Holding the vial slightly tilted, use a sterile loop to transfer one loopful (10ul) to the +DNA tube. (The loop is designed to hold 10 ul of fluid when the fluid forms a complete bubble across the loop opening.) Recap the tube and return to the ice bath. DO NOT ADD PLASMID TO THE “-DNA” TUBE. Mix and allow both tubes to stand on ice for 15 minutes. The competent cells will now begin to take up the plasmid DNA. Place both tubes in a 42 degree C water bath for 90 seconds to heat shock the cells. Return the tubes immediately to the ice. (An extended period of time on ice at this point in the experiment will not affect the outcome.) Remove tubes from ice and place them in test tube rack. Use a sterile pipet to add .25 ml (250ul) Luria Broth - and a like volume to the +DNA tube. Gently tap tubes to mix the contents. Incubate at 37 degrees C for 10 minutes. This incubation period allows the cells to recover from the Ca012 treatment and begin to express antibiotic resistance. Due to their small size you may wish to place tubes in a small beaker before placing in the incubator. While waiting. obtain 1 LB plate and 1 LB+amp plate. Use a marker to draw a line on the back of each plate dividing the plate in half. Label one side of each plate '+' and one side '-“. 73 x11. Remove the tubes from the incubator. Use a sterile pipet to transfer .1 ml (Figure 1) from “- DNA" tube to the “-“ sides of both plates, and transfer .1 mi from '+ DNA“ tube to the “+“ sides of both plates. '- “ first; Flame an inoculating loop. allow it to cool sufficiently. and streak the solution in a zig-zag fashion on the appropriate side the each plate (Figure 2). Flame and cool inoculating loop each time. 12. Let the plates sit in a designated area. Your teacher will invert the plates and incubate at 37 C overnight. A successful transformation will be indicated by the appearance of colonies on both sides of the LB plate, and on the "+'I side of the LB+ampicillin plate, and by absence of colonies on the '-” of the LB+ampicillin plate. ANALYSIS AND DISCUSSION: 1. Where did colonies appear? 2. Explain the reason or reasons for the presence or absence of growth on these plates. 3. How do you know that the plasmid is present in the transformed cells? 4. If this was part of a recombinant DNA experiment, what additional steps would have been included? APPENDIX B TEACHER’S GUIDES TO LABORATORY EXERCISES APPENDIX B TEACHER’S GUIDES TO LABORATORY EXERCISES TEACHER’S GUIDE TO EFFECT OF BUFFERS ON THE pH OF A SOLUTION W After the laboratory acitivity is completed. the student will be able to correlate the concept of buffering action to the maintenance of a selected pH. The student will be able to record. graph. and interpret data generated by laboratory activity. W Igggh§;_gggg§;§;ign: The .1 M NaOH. .1 M HCi. and pH buffer solution should take no more than 1 hour to prepare. Student_lab_&1me: The lab will take less than fifty minute class period. MW: 1. Prepare 0.1 M solution of NaOH by dissolving 4 g of solid NaOH in 100 mL of distilled water. 2. Prepare 0.1 M solution of HCl by adding 1 mL of concentrated HCl to 100 mL of distilled water. 3. Prepare pH 7 buffer. W: The control of an almost constant pH in living systems is achieved by buffers. A buffer is a substance capable of neutralizing both acids and bases in a solution and maintaining close to the original pH of the solution. 74 75 Limestone is mainly calcium carbonate which is a salt. Carbonates and bicarbonates of calcium act as buffers in natural waters and some soils: that is they help to stablize the pH when acids or bases are added. Of course. when large amounts of strong acids or strong bases are added. the buffering capacity may be exceeded and the pH may change enough to kill organisms. The breakdown of carbonates and bicarbonates releases carbon dioxide that aquatic plants use in photosynthesis. The most biologically productive lakes are often quite alkaline. The soils of lower peninsula of Michigan have more limestone base than the upper peninsula. Canada and northeastern United States. Therefore. the lower peninsula of Michigan has less of a problem with acid rain. Stomach antacids are usually calcium carbonates. W3 1. The pH of the distilled water went down as HCl was added. 2. The pH of the distilled water went up as NaOH was added. 3. The pH remained nearly constant. 4. Answers may vary. but should indicate the need for living things to maintain nearly constant internal pH. 5. Limestone is calcium carbonate which acts as a buffer in soils and lakes. 6. The pH would be lowered by acid rain, possibly adversely affecting the living organism. 76 TEACHER’S GUIDE TO TESTING FOR ORGANIC MOLECULES BEHA!IQRAL.QBJE§II¥ES* The student will be able to correlate the color changes of Benedict’s solution with the presence of glucose. The student will recognize that sucrose will not react with Benedict’s solution. The student will be able to correlate iodine’s blue-black color with the presence of starch. The student will recognize that a translucent spot on brown paper indicates the presence of lipids. The student will recognize that when oil and water are mixed. Sudan will stain oil a pink color. The student will recognize that Biuret reagent changes from blue to a faint purple color in the presence of protein. The student will be able to perform the above test on unknown food samples to determine the presence of sugar, starch. lipid and protein. W: Iggghgz_nggnggggign: The solutions will take about an hour to prepare. Some the reagents may be purchased ready made from supply houses such Carolina Biologicals. These would include Benedict’s solution. iodine solution. Sudan. and Biuret reagent. : The lab will take two class periods. Many students will complete most the tests of known solutions the first day. and test unknowns the second day. Some the student groups will need part of the second day to finish the tests of known solutions. W: 1. Using a soluble starch solution such as Baker’s Analysed potato starch or arrowroot. make a 1% starch solution by dissolving 5 g of potato starch in 500ml of distilled water. Heat to boiling. stirring occasionally. Allow solution to cool to 77 stirring occasionally. Allow solution to cool to room temperature before using. Boiling the starch solution will prevent settling in the test tube. To prepare Lugol’s iodine solution. dissolve 3 g of potassium iodide (KI) in 25 mL of water. Then add 0.6 g of iodine crystals, and stir until dissolved. Make a 1:10 dilution of this stock. Store stock in a dark bottle. W Thoe positive test results are given in the directions. Candy bars, cookies, and other sweet foods usually contain sucrose. Sucrose is not a reducing sugar and will not react with Benedict’s solution. Onions store carbohydrates in the form of simple sugars: potatos store carbohydrates in the form of starch. W: Most biological supply houses carry the needed materials. 78 TEACHER’S GUIDE TO AFRICAN VIOLET TISSUE CULTURE LAB WES: At the end of this activity. the student will be able to define cloning. tissue culture. nutrient media. totipotency. callus. differentiation. plant hormones. and sterile technique. The student will gain practice in using sterile techniques The student will recognize the need for accurate record keeping since so much time elapses between the beginning and the conclusion of excercise. WED: Ieagbgn_pneganntign: The preparation for this lab can be time consuming. but the prepared culture tubes can be purchased ready made from Carolina Biological and other supply houses. The materials can purchased from supply houses and prepared and sterilized. They may also be purchased from Howard Hagerman. Lyman Briggs. Michigan State University. stgggn;_lgp_;img: One day is suggested to demonstrate the entire procedure and prepare students. The lab itself can be preformed in one fifty or fifty five class period. if students are prepared. After cultures are started, students will need about 10 minutes once a week to record observations. W: 1. Decide how many groups you will be working with. Fill a 250 mL beaker or flask half full with distilled water for each group. Cover mouth of beaker or flask tightly with foil. 2. Cover the top of enough clean empty 250 mL beakers tightly with foil. 3. Wrap a scalpel, a pair of long forceps and a pair of shorter forceps in foil. This step can be avoided by dipping forceps in alcohol and passing through a flame Just prior to use. 79 4. Sterilize beakers. flasks and other equipment using pressure sterilizer. 5. The Murashige-Skoog medium that was used were purchased from Howard Hagerman. Michigan State University. Each group had a set of four tubes with the following contents: 1. 0.18 IAA and 0.2 Kinetln 2. 3.00 IAA and 1.0 Kinetin 3. 0.0005 IAA and 0.2 Kinetin 4. control - no hormones Since veins contain some hormones, it is best to try and avoid them in the cultures if at all possible. If there are maJor veins in culture. it will be difficult to tell if growth is from the hormones in the medium or the veins. The students should have a thorough background in sterile technique. You might want to have them practice the techniques several times before doing the actual lab. Sterility cannot be stressed enough. Low success rate can be very discouraging for the students. A 50% success rate can be considered pretty good. When the cultures finally grow both roots and shoots (it takes 4 to 8 weeks), you can transplant them to sterilized potting soil. Use a long thin spatula to loosen the media. Hold the tube upside down and gently tap the edge. Gently wash the media off the roots before transplanting. The plant and container will have to be covered with plastic to prevent it from 80 drying out. After it begins to grow. gradually remove the cover or Just poke some holes in the plastic. W: 1. Carolina Biological Supply House (2700 York Road. Burlington. North Carolina 27215) has the best selection of tissue culture supplies. 2. Ward’s Natural Science (5100 West Henrietta Road. PO Box 92912. Rochester. New York 14692-9012) has limited tissue culture supplies. 81 TEACHER’S GUIDE TO LAC OPERON BEHA!ORLAL.QBJE§IL¥ES¢ The student will be able to inoculate a bacterial culture using sterile technique. After completion of this lab the learner will be able to recognize that all messenger RNA is not transcribed all of the time. After completion of this lab the learner will be able to define and use in context the following terms: operon. operator. promoter. structural gene, regulator gene, repressor protein. and RNA polymerase. The learner will recognize the importance of lactose in the induction of lactase production in E. coli. IIUE_REQULRED= W: This lab requires the preparation and sterilzation of culture media. Approximately an hour would be necessary to prepare the glucose and lactose culture media and O-NPG solution. Additional time would be necessary for sterilzation of media and preparation of E. coli. W1: Approximately twenty to thirty minutes would be required the first day to inoculate cultures. The teacher should allow time before lab to demonstrate inoculation of cultures. Approximately thirty minutes would be needed the second day to add O-NPG and make observations. MAIERLAL.RRERARATLON: 1. Culture media a) Prepare 900 ml of stock solution. Half of this will be used to prepare lactose broth and half used to prepare glucose broth. NH4CI 1 g Na2HP04 6 9 KH 04 3 9 Na I 5 g MgSO4 0.1 g distilled water 900 mL 82 b) Prepare 0.5% sugar solutions. 0.5% lactose 0.5% glucose lactose .5 g - glucose 0.5 g distilled water 100 mL distilled water 100 mL c) Lactose broth: To 450 ml of stock solution. add 50 mL of 0.5% lactose solution, producing 500 mL total of 0.05% lactose broth. Glucose broth: To 450 mL of stock solution. add 50 mL of 0.5% glucose solution.producing 500 ml total of 0.05% glucose broth. Pour this into culture tubes. cap and sterilize. (Each culture tube holds approximately 20 mL of broth. Each team needs one lactose broth and one glucose broth) d) 0.5% O-nitrophenyl-beta-D-galactopyranoside (O-NPG): Add 0.5 grams O-NPG to 100 Ml distilled water. DAQKGRQUND_INEQRMAILQN3 Lactose sugar induces the production of lactase (B-galactosidase) by binding with the repressor protein thus inactivating it. This allows the RNA polymerase to bind to promoter which allows the structural genes to produce RNA for lactase. and two other proteins. Lactase is shown to be present by reaction with O-NPG. Both O-NPG and lactose are substrates for the enzyme. When O-NPG is cleaved by the enzyme, a yellow substance appears. It is the appearance of the yellow color, after twenty hours of incubation and addition of O-NPG that verifies the production of lactase. The glucose broth will not produce lactase and therefore. not produce the yellow color. 83 Expected results: Legend: + astrongly yellow: - a weakly yellow: 0 a no color 10 min. 20 min. 30 min. Lactose - + + Glucose 0 0 0 Carolina Biological has the materials in a “Gene Regulation Biokit“. This would be more expensive but also more convenient. especially if no autoclave is ' available for sterilizing media. W: Stryer. L. (1981) Bigghemistny. New York: Freeman and Company. Howard Hagenman. "Effect of Lactose on Lac Operon: A Study in Gene Regulation“ Michigan State University. Adapted from Carolina Biological Gene Regulation Biokit. 84 TEACHER’S GUIDE TO TRANSFORMATION OF BACTERIA Note: The student laboratory was tested and found that the procedure could be adapted to the high school class period of approximately 50 minutes. When ordering materials. it was found that materials could be purchased most economically by purchasing the “Colony Transformation Kit“ from Carolina Biological. By making a few adapations. the kit served 3 classes (the two classes in this study and a Microbiology class). One adaptation was ordering additional Luria Agar and Luria Agar with Ampicillin for plating the bacteria. The plasmid in the kit may not be pBR322. Plasmid pBR322 was used when the lab was tested. and does contain the ampicillin resistant gene. SOURCE_OE_MAIER1ALS 1. Carolina Biological Supply House (2700 York Road. Burlington. North Carolina 27215) APPENDIX C DETAILED OUTLINE OF LESSONS APPENDIX C DETAILED OUTLINE OF LESSONS MODULE I Part 1 Introductory Chemistry DAY 1 INTRODUCTORY CHEMISTRY W 1. Describe the structure of an atom. using the terms proton. neutron. electron. atomic number and shell. 2. Define valence and give valences of hydrogen. oxygen carbon and nitrogen. 3. Describe a covalent bond. Materials; 1. Pre-test 2. Overhead transparency of: A. Periodic Table B. Covalent bonds of the water molecule 3. Worksheet “Atoms and Chemical Bonding“ Procedures. 1. Administer pre-test. 2. Provide a definiton for terms proton, neutron, electron. atomic number. shell. valence. and covalent bond. 3. Draw the atoms of Hydrogen. Oxygen. Carbon and Nitrogen on the board to illustrate valence. 4. Provide a definition of molecule. molecular formula. structural formula. single covalent bond. double covalent bond, and triple covalent bond. . Use the example of the water molecule to illustrate the concepts of molecular formula, structural formula and covalent bond. 6. Handout worksheet on atoms and chemical bonding. Assign for homework. Wei: 1. Note that preceding this module. six class periods were used to instruct students on process skills. 85 86 Various activities were used to cover the process skills of controlling variables. hypothesis writing. making data tables and making graphs. Throughout the year course. process obJectives are important as well as content obJectives. Below is a complete list of process obJectives covered during the year. All tests given through the year test both process and content obJectives. The first test of the year. given on day seven of the module. tests for process obJectives 1. 2. 3. 5. 6. 7. 11. 14. 15, 17, and 18. It is understood by the students that once a process obJective is introduced it will be used and tested at any time during the course. PROCESS OBJECTIVES I. II. III. Formulating Hypotheses 1. Distinguish between hypotheses and statements which are not useful as hyptheses. 2. Identify data which support stated hypotheses. 3. Formulate hypotheses. 4. Identify or design tests of hypotheses. Controlling Variables 5. Distinguish between variables which can be manipulated and those which cannot. 6. Identify the variable(s) that should be held constant during the course of a laboratory exercise. Interpreting Data 7. Identify the component parts of scientific laboratory exercise. control group. experimental groups. etc. 8. Identify new testable hypotheses based on the results of laboratory exercises. 9. Formulate a question or define a problem to be tested on a laboratory exercise. 10. Given a laboratory problem and hypothesis. select suitable materials and design 87 appropriate procedure for testing hypothesis and appropriate observation methods. IV. Interpreting Data 11. 12. 13. Examine the data collected in a laboratory exercise and select the sets of data which appear to be related. Construct generalizations from sets of data. Identify the data or diagrams to convey information collected during laboratory exercise. V. Communication 14. 15. 16. Construct a data table for organizing data collected during the laboratory exercise. Construct bar and line graphs of investigative data. Make drawings or diagrams to convey information collected during laboratory exercise. VI. Predicting 17. 18. 19. Interpolate from graphs or data. Extrapolate from graphs or data. Identify or design tests for predictions made from experimental data. VII. Inferring 20. 21. Identify or construct reasonable explanations of observations. Use references to support explanations of observations. DAY 2 CHEMICAL MODEL BUILDING Obieetixes 1. To define valence and apply the concept of valence to chemical model building. 2. To define covalent bond and apply the concept to chemical model building. Metering 1. Chemical Model Building Kit from Carolina (Packaged into one set for each pair of students) 2. Worksheet 3. Overhead transparency of carbon skeletons. Rnpeedime 1. Handout worksheet and chemical model building kits. Instruct students to produce a model for each of nine formula being sure to follow the laws of valence. Explain that carbon atoms form various carbon skeletons. A. Use overhead transparency. 8. Advice students to begin by Joining carbon atoms together. Instruct students to draw on worksheet a two dimensional drawing of their model. Produce a model of the first molecule using one carbon atom and four hydrogen atoms. A. Have students work in groups of two. B. Have all student groups produce the same model. Draw the molecule on the board and discuss. C. Point out that the molecule produced is called methane. WM Most student groups have some difficulty with the double bond. They can more easily accept the double between carbons than between carbon and oxygen as in the carboxyl group. The students appear to enJoy this activity. 89 DAY 3 pH - ACIDS. BASES. AND INDICATORS W 1. Provide a definition for the terms ion. ionic bond. 2. Show how ionic compound dissolves in water compared with how covalent compound dissolves in water. 3. Define acid. base. hydrogen ion. and hydroxide ion. 4. Explain pH. pH indicator. neutralization. and salt, while demonstrating the use of pH indicator bromthymol blue. W 1. Overhead transparencies of: A. sodium atom. chlorine atom. sodium ion. and chlorine ion B. crystal of alternating sodium and chloride ions C. Sodiumland chloride ions dissolved in water D. Comparison of ionic and covalent solution E. Formation of an acid in water F. Formation of a base in water 2. Student handouts of diagrams used on overhead transparencies. 3. Materials for 'Blue and Gold“ Demonstration A. .1M HCl B. .1M NaCl C. bromthymol blue solution D. pH hydrion paper and/or electronic pH meter E. beakers Procedure 1. Collect homework from two previous days- “Atoms and Chemical Bonding“ and worksheet on Chemical Model Building. 2. Review concept of valence from previous two days. 3. Define an ion as an atom that has gained or lost one or more electrons. A. Remind students that valence is the number of electrons that the atom must share. lose or gain to complete its outer shell. 10. 90 Point out the difference the covalent bond of the water molecule and the ion bond of sodium chloride. EXplain the polar character of water. A. Defin Use the overhead transparency to show how sodiwm chloride dissolves in water to form sodium ions and chloride ions. Use the overhead transparency to show how glucose sugar dissolve in water. e dissociation and use water as an example. Define acid as a substance that releases hydrogen ions A. B. C. in water. Use HCl as an example. Draw beaker of water on board to show dissociation of HCl. Use handout and overhead transparency. Define base as a substancae that releases hydroxide ions A. B. C. in water. Use NaOH as an example. Draw beaker of water on board to show dissocation of NaOH. Use handout and overhead transparency. Define pH and draw a simple pH scale on the board. A. B. C. D. If the number [concentration] of H+ is greater than the number of OH-. the solution is acidic or acid. Define pH indicator. Use bromthymol blue as an exanple of pH indicator. Bromthymol blue turns yellow or green in acid and blue in base. Demonstrate use of bromthympl blue in "Blue and Gold' A. demonstration. Fill three beakers with approximately 100 ml of water. Pour .1M HCl into one beaker. nothing in second beaker, and .1M NaOH into third beaker. Ask students to predict color changes as bromthymol blue is poured into each beaker. Base will turn blue. and the acid will turn yellow (gold): thus the “Blue and Gold“. Define neutralization. 91 A. Pour acid and base together from demonstration: a neutral blue-green will be produced. B. Introduce a balanced equation by writing equation for the reaction: HCL + NaOH ---> NaCl + H20 ( acid + base --> salt + water) We 1. The difference between the covalent bond and the ionic bond is often a difficult concept for students. This is a good time to explain the difference between a molecule and a compound: however. this was not a concept that all students understood. 2. The school colors are blue and gold. therefore. the demonstration has been called the “Blue and Gold“ demonstration. When bromthymol blue is used later in the course, as in the detection of carbon dioxide in respiration. the students remember the 'Blue and Gold'' demonstration. 3. Some students are able to accept the view of a base as a hydrogen acceptor. Most find this too difficult. DAY 4 Laboratory Exercise: DISCOVERY OF pH OF USING COMMON HOUSEHOLD SUBSTANCES Dbleetixes 1. To demonstrate that indicators can be used to determine pH of a substance. 2. To familiarize students with the fact that different items have a different pH. 3. To familiarize students with the use of a data table. Materials 1. See lab for list of lab materials: 2. Lab Report Outline - a standard form to be used for almost all lab reports in this class. W Have students predict the pH of at least one unknown solution and record this on the lab report under hypothesis. Students use samples of diluted materials such as. orange Juice. coffee. shampoo. ammonia. soft drinks and other materials found around the home. The samples are tested using a pH indicator strip. The pH is recorded in table. Students are instucted to make a copy of the data table on their Lab Report Outline. EXplain how the Lab Report Outline should be completed and assign its completion for homework. W This is a very simple laboratory session. It is very appropriate for the first lab of the year. The students seem to enJoy using the pH indicator strips and especially find testing their soft drinks interesting. The lab is completed with enough time remaining to explain how the Lab Report Outline is to be filled out. This type of lab report will be expected following all laboratory exercises. It is to be completed for homework and turned in at the beginning of the next class period. If questions are included at the end the laboratory exercises. these are usually discussed at the end of the lab period and it often suggested that information from the answers to these questions be included in the lab report. DAY 5 Laboratory Exercise: EFFECT OF BUFFERS ON THE pH OF A SOLUTION W 1. To define buffer and homeostasis. To relate pH to acids. bases and salts. To relate the concept of pH to the buffering action and the maintenance of a selected pH. 93 4. To express buffer action in terms of homeostasis in living systems. 5. To record data in data table. 6. To graph data. identifying independent from dependent variable. MW 1. See lab for lab materials: 2. Lab Report Outline with grid for graph 3. Carolina Tips I'Acid Rain:The Bitter Dilemma" Procedure 1. Collect Lab Report for "Discovery of pH Using Common Household Substances'I and discuss briefly. 2. Define buffer as a substance that keeps pH constant by taking up or releasing H1’ or 03- as these are added to the solution. 3. Discussion of the material written in the introduction to the lab. stressing the importance of maintaining a constant internal environment. 4. Ask for two volunteers, one to read the pH paper as HCl and NaOH is added to distilled water. and one to read the pH paper as HCl and NaOH is added to buffer. 5. Give time for students to answer the questions at the end of the lab. Remind students to read the section on homeostasis in their text book. 6. Discuss the questions at the end of the lab. 7. Instruct students to complete lab report by including a copy of the data table. completing the graph. properly labeling the graph. and including a discussion of the answers to the questions in the conclusion. 8. Assign reading of Carolina Tips 'Acid Rain:The Bitter Dilema' for homework. W21“: 1. This lab requires 40 strips of pH paper: 20 for the distilled water and 20 for the buffer. If pH 94 meters or sufficient pH paper is available. this lab could be performed by all student groups. 2. This lab provides the first lab data to be graphed. Many of the labs throughout the year will require that the student determine the independent and dependent ' variable and properly graph data. DAY 6 ACID RAIN QDJEQSllfifl 1. To determine the sources of pollutants responsible for acid rain. 2. To determine the consequences of acid rain. 3. To recognize that limestone in the soil can have a buffering effect for acid rain. M12131: 1. Carolina Tips 'Acid Rain:The Bitter Dilemma“ 2. Worksheet “Introductory Chemistry“ EIQQIQHZE 1. Collect lab report for "What is the Effect of Buffers on the pH of a solution?“ 2. Discuss the importance of buffers in living cells and the concept of homeostasis. 3. By means of lecture and reference to the reading of Carolina Tips article. discuss the sources of sulfuric acid and nitric acids in acid rain and the effects on living things. 4. Ask the students to read the Carolina Tips again. this time identifying the “bitter dilemma“ referred to in the article. 5. Assign “Introductory Chemistry Worksheet“. A. When students have finished. go over answers and have students correct there own in class. Netes_and_aneedptai_mateniaie 1. Most students do not read the Carolina Tips article when it is first assigned. It takes a while for students to realize that video tape program and 95 outside reading of the type used here are often sources of essay questions on tests. DAY 7 VIDEO TAPE 'Acm RAIN' museum: 1. To determine the sources of pollutants responsible for acid rain. 2. To determine the consequences of acid rain. 3. To recognize that limestone in the soil can have a buffering effect for acid rain. MW 1. Video tape of Nova’s "Acid Rain" 2. Worksheet of questions W 1. Show video tape. 2. Require students to complete questions on worksheet while viewing video. Wm 1. Students are usually not as impressed by the effects of acid rain on the lakes and streams as they are by the effects on the forests. DAY 8 DISCUSSION mm: 1. Describe the structure of an atom. using the terms proton, neutron, electron. atomic number and shell. 2. Define valence and give valences of hydrogen, oxygen and carbon. 3. Describe a covalent bond. 4. Explain what an ion is. and how it forms. 5. Describe an ionic bond. 6. Describe the polar properties of water and explain how this affects ionic compounds. 7. Explain what is meant by pH. 8. Define acid and base. 9. Specify the pH of the material within most living cells and indicate whether this is acidic. neutral or basic. Identify buffers as compounds that resist changes in pH. 10. Define homeostasis and explain how buffers help maintain homeostasis. Manuals 1. Overhead transparencies used in module. Procedure 1. Collect worksheet on Acid Rain video and discuss. 2. Return all corrected papers that have not yet been returned. 3. Review all labs and relate to concepts and process skills that have been covered in module. DAY 9 TEST MODULE I Part 2 ORGANIC CHEMISTRY DAY 10 PRE-TEST. CARBOHYDRATES W 1. Define the following terms and use in context: Organic molecule. inorganic molecule. molecular formula. structural formula. carbohydrate. monosaccharide. disaccharide. polysaccharide. 2. Name the three types of organic molecules. 3. Know the uses of the organic molecules in the cell. 4. Name the three types of carbohydrates and examples of each. MALQELALS 1. Handout “organic molecules” 97 2. Handout “Biological Roles of Various Organic Molecules“ 3. Overhead transparency of monosaccharide structural formulas. Procedure 1. Handout packet of materials that includes lecture obJectives and diagrams to supplement textbook. 2. Provide definition. explanation and examples of the following: Organic molecule. inorganic molecule. molecular formula. and structural formula. 3. Give examples of carbohydrates. 4. Discuss examples of monosaccharides. disaccharides. and polysaccharides. 5. Discuss specific uses of monosaccharides. disaccharides. and polysaccharides in the cell. 6. Inform students of quiz the next naming example of monosaccharides. disaccharides. and polysaccharides We 1. Student backgrounds can vary considerably. Some students know the names of the carbohydrates and their roles in cells. many claim that they have never heard of them. ‘DAY 11 DEHYDRATION SYNTHESIS AND HYDROLYSIS OF CARBOHYDRATES W 1. Define the following terms and use in context: Organic molecule, inorganic molecule. carbohydrate. monosaccharide. disaccharide. polysaccharide. dehydration synthesis. hydrolysis. and hydroxyl group. 2. Name the three types of carbohydrates and examples of each. 3. Show the dehydration synthesis and hydrolysis of carbohydrates. Manuals 98 1. Short quiz over obJective 2. 2. Overhead transparencies of: A. monosaccharide structural formulas B. dehydration synthesis and hydrolysis of maltose C. polysaccharide structures 3. Organic Molecule WOrksheet pages 1 and 2. Procedure 1. Administer quiz. 2. Review terms and concepts from previous DAY. 3. Explain dehydration synthesis of maltose. 4. Assist individual students in completion of dehydration synthesis of maltose on top of page two of worksheet. 5. Explain hydrolysis of maltose. 6. Assist individual students in completion of hydrolysis of maltose on page two of worksheet. 7. EXplain dehydration synthesis and hydrolysis of polysaccharides with emphasis on amylose. 8. Assign completion of pages one and two of worksheet. Notee_ano_aneooota1_mater1a1s 1. Most students need some assistance in completing the worksheets. DAY 12 LIPIDS leeetixes 1. Define the following terms and use in context: dehydration synthesis. hydrolysis. lipids. glycerol. fatty acid. saturated fatty acid. unsaturated fatty acid. carboxyl group. and hydroxyl group . Know the uses of lipids in the cell. Show the dehydration synthesis and hydrolysis of lipids. 99 Materials 1. Overhead transparency of: A. dehydration synthesis and hydrolysis of lipids. B. saturated and unsaturated fatty acids 2. Organic Molecule Werksheet pages three and four Procedure 1. Discuss the composition of lipids and their role in cells. 2. Discuss the structure of glycerol. 3. Discuss the general structure of a fatty acid. 4. Explain dehydration synthesis and hydrolysis of lipids. 5. Assign completion of page three and top of page four. DAY 13 PROTEINS Dbleetixes 1. Define the following terms and use in context: dehydration synthesis. hydrolysis. carboxyl group. hydroxyl group. protein. amino acid. amino group. and peptide bond. 2. Know the uses of protein molecules in the cell. 3. Show the dehydration synthesis and hydrolysis of proteins. 4. Explain how amino acids differ from one another. Materials 1. Overhead transparency of: A. dehydration synthesis and hydrolysis of dipeptide. B. twenty amino acids 2. Organic molecule worksheet page five and six. Procedure 1. Discuss the composition of proteins and their role in cells. 4. 100 A. Emphasize that a change in order and number of amino acids changes the function of protein. Explain general structure of amino acid. A. Use transparency same as handout. 8. Discuss significance of R group. Explain dehydration synthesis and hydrolysis of dipeptide and fonmation of peptide bond. Assign completion of worksheet pages five, six and the bottom of page four. DAY 14 PROTEINS AND ENZYMES W 1. Know the uses of protein molecules in the cell. 2. Explain how amino acids differ from one another. 3. Be able to tell whether the R group of an amino acid is charged and identfy which charge it would have in an aicd solution and which charge it would have in a basic solution. 4. Define enzymes and substrate. 5. Explain why the three-dimensional structure of an enzyme is the key to its activity. In doing so. include answers to following questions: What is the active site of the enzyme? Why may changes in temperature or pH greatly reduce enzyme activity? Materials 1. Overhead transparencies A. primary structure and conformation of insulin and myoglobin 2. Rubber tubing 3. Two slinkies. one new. the other badly twisted. 4. Filmstrip “Enzymes“ 5. Worksheet “Enzyme Structure and Function“ to be completed while watching filmstrip. Procedure 1. Draw and explain the lock and key model of enzyme action. 101 2. Explain the naming of enzymes. using amylase. maltase and sucrase as examples of carbohydrate hydrolysis enzymes. 3. Explain the signficance of sulfur containing amino acids in the primary structure of the polypepide. A. Use the rubber tubing to demonstrate the three dimensional structure could be formed by disulfide bonds. 4. Explain the relationship between the structure of enzyme. its conformation, and the function of the enzyme. A. Use the new slinky to show its conformation allows it to “work“. that is. climb down a staircase of books. 8. Use the badly twisted slinky to show how denaturing of a protein changes its shape and does not allow it to “work“. WM 1. It was decided not to discuss other factors leading to conformation changes. The classes had very good questions about structure / function relationships following the slinky demonstration. DAY 15 Laboratory Exercise: TESTING FOR ORGANIC MOLECULES ghlflgillflfl 1. To perform and interpret the results of the Benedict’s test for reducing sugar. 2. To perform and interpret the results of iodine test for starch. 3. To perform and interpret the results of the Sudan III test and Brown Paper test for lipids. 4. To perform and interpret the results of the Biuret test for proteins. 5. To use the specific chemical tests to determine the presence of sugar. starch. lipid, and protein in UNKNOWN FOOD SAMPLES. 11mm: See lab for lab materials 102 Procedure 1. Instruct students briefly in the use of Benedict’s solution. iodine solution. Brown paper test and Biuret reagent. 2. Provide known solution to be tested. WM 1. Most students complete at least three tests the first day. They ususally complete the Benedict’s test. iodine test. and at least one the tests for lipids. Very few groups complete the Biuret test. 2. Many students do not take seriously the caution concerning boiling solutions in a test tube. This is a good time to emphasize lab safety. DAY 16 Laboratory Exercise: TESTING FOR ORGANIC MOLECULES continued W 1. To use the specific chemical tests to determine the presence of sugar. starch. lipid. and protein in UNKNOWN FOOD SAMPLES. Materials Lab materials EEQQEQMER 1. Briefly summarize and discuss results of tests from previous day. 2. Identify list of unknowns available. including any materials brought in by students. 3. At end of class. discuss the results of unknown test. Not all students will finish testing all materials. Notes.and_aneedote1_mater1ale 1. Most students need the second day to complete the Biuret test for proteins. and all groups need this second day to complete tests of unknown foods. 103 DAY 17 EFFECT OF pH ON PROTEIN DIGESTION BY PAPAIN Quentin: 1. To follow the enzymatic breakdown of a protein. 2. To determine the effect of pH on enzymatic action. Materials 1. Lab Materials See lab for lab materials 2. Lab Report Outline Procedure 1. Briefly remind students of discussion of effect of - pH on conformation of enzyme. 2. Ask students to write a possible hypothesis on lab report outline. 3. Briefly discuss lab procedure. Notes_and_anec.do.tai_materiaie 1. Any acid or base solutions will work. .1M HCl and .1 M NaOH. for example. would work fine. 2. Knox gelatin was prepared using half the necessary water. Unfortunately it did not digest during the lab period. It has subsequently been found that Jello brand gelatin poured in a thin layer works best. DAY 18 INTRODUCTION TO SPECTROPHOTOPMETRY AND PIPETTES leeetIuee 1. To relate the principles of the behavior of light that are used by spectrophotometers to human optical perceptions. 2. To relate the concentration of an iodine-starch solution to the percent light transmitted. 3. To become familiar with the use of a spectrophotometer. 4. To gain experience using a pipette by pipetting colored water. 104 5. To gain experience using a pi-pump. Materials Overhead transparency of parts of spectrophotometer test tube Low concentration of iodine-starch solution High concentration of iodine-starch solution Spectrophotometer 1 ml and 5 ml pipettes Blue and green pi-pumps Water with food coloring for practice pipetting Procedure 1. Use overhead transparency and refer to diagram on student handout that shows the relationship among the following parts of the spectrophotometer: light source. monochromatic filter. monochromatic light. tube (cuvette). photocell and transmittance scale. 2. Use the example of blue color produced by iodine-starch solution to explain the the use of the wavelength control knob. 3. Use overhead transparency to explain 100% transmittance of light using blank and reduced transmittance of light with dark blue iodine starch solution. 4. Use two different concentrations of iodine starch solution and ask students to predict which one would give higher transmittance reading. 5. Suggest that amylase. which digests starch. is added to test tube of high concentration iodine starch solution. A. Have students predict what would happen. B. Explain that amylase digesting starch and changing the intensity of starch solution is exactly what the next day’s lab will be doing. 6. Demonstrate the use of pi-pumps and pipettes 7. Let students practice using pipettes to pipette colored water. 105 NoreeJndJneddoraLmaterLaLe 1. This lesson on the use of spectrophotomer such as the Spec 20 does not concentrate on training students how to adJust and zero in the instrument. The purpose of the lesson is to relate the concentration of an iodine-starch solution to the percent light transmitted. 2. This lesson also is not intended to be the only exposure to the concept of the standard curve. Two days will be spent on a lab designed to use the standard curve. 3. The students enJoy using pipettes and pi-pumps. DAY 19 Laboratory Exercise: EFFECT OF TEMPERATURE on the ACTIVITY OF SALIVARY AMYLASE W 1. To discover some of the physical changes that occur during starch digestion. 2. To become familiar with the use of the Spec 20 or other colorimeter. 3. To measure the effects of temperature on the speed of starch digestion. 4. To record and graph data. Materials 1. See lab for Lab materials 2. Overhead transparency of parts of spectrophotometer 3. Lab Report Outline with grid for graph Procedure 1. Use overhead transparency to review relationship of the concentration of an iodine-starch solution to the percent light transmitted. and to review use of spectrophotometers. 2. Pass out Lab Report Outline . A. Ask students to hypothesize what will happen to the starch concentration at different temperatures. B. Ask students to record their hypothesis on Lab Report Outline. 3. 4. 5. 106 Assign lab groups of four. Briefly explain procedure for lab. Demonstate how to blank the spectrophotometer. but emphasize that this has been done for them. WM 1. Students should begin timing as soon as saliva is added. Careful timing and reading will lead to the most accurate data. 2. Test tube A was used as a control. B at room temperature. and C in ice water. Test tube 0 in warm water would not really be necessary to show the effect of temperature on rate of enzyme reaction. and was not used here. 3. Two spectrophotometers were used with six groups of four students. By eliminating the test tube in warm water. two spectrophotometers served the whole class. DAY 20 Lab: DETERMINING CONCENTRATION OF PROTEIN IN MILK Obieotixee 1. To determine the concentration of proteins in milk. 2. To gain experience using a pipette. 3. To gain an appreciation of the use and operation of the spectrophotometer. 4. To make and interpret a graph. 5. To gain experience using a standard curve of known protein concentrations to interpolate the concentration of an unknown protein. Materials 1. See lab for Lab materials 2. Lab Report Outline with grid for graph 3. Computer program “Analytical Graph“ 107 Procedure Collect the lab report for “Effect of temperature on the activity of salivary amylase“ A. Discuss the previous day’s lab. B. Coupare the usage of spectrophotometer in previous day’s lab and today’s lab. Explain to students that the first day will concentrate on data collection and the second day will concentrate on graphing and interpreting data. Explain procedure for the lab. Assign students to same group of four as in previous lab. Obtain a copy of the data from a typical group to be plotted using the computer program “Analytical Graph“. Suggest to students that they could attempt to plot the results of the first six test tubes. but there would be time in class the next day. WM DAY An overhead transparency is made from the data plotted on “Analytical Graph“ and used the next day to explain the use of a standard curve. Student confidence in the use the spectrophotometer increases with the second use of the instrument. Some students begin to show an interest in knowing how to zero in the instrument and other possible uses of the instrument. The full sixty minute class period was needed to complete readings. but all groups did finish. For a shorter class period. another day might be needed. I 21 Lab: DETERMINING CONCENTRATION OF PROTEIN IN MILK (continued) W 1. To make and interpret a graph. 108 2. To gain experience using a standard curve of known protein concentrations to interpolate the concentration of an unknown protein. 11mm: 1. Overhead transparency of data from lab on previous day plotted on “Analytical Graph“. 2. Lab Report Outline with grid for graph. Proeedure 1. Introduce the concept of standard curve. A. Suggest that four known concentrations of starch solution were measured in the spectrophotometer. B. Draw graph on the board with the fictional readings from the spectrophotometer. C. Identify that this graph of known concentration would be called a standard curve. D. Now suggest that an unknown concentration was read in the spectrophotometer: using interpolation or extrapolation determine the concentration of the unknown. 2. Stress that there are many ways to use the spectrophotometer in research. A. One use of spectrophotometry is determining the rate of enzyme activity. as in the first example of iodine-starch solution with amylase. B. Another use of spectrophotometry is determining the concentration of a solution. as in the second example with the standard curve. 3. Use the overhead transparency of data plotted on “Analytical Graph“. A. Only the known concentrations should be plotted. B Place an “X“ on the line where the reading of the unknown, the diluted milk sample. would intercept the graph. C. Using interpolation. estimate the concentration of protein in the milk. 4. Assist students in completing their own graphs and estimating the concentration of protein in their milk sample. 5. 109 Discuss the class results Noted_and_aneedotal_materi.ale In determining the concentration of protein in the milk sample. be sure that the students correct for the dilutions. The number that they get from the spectrophotometer is the concentration in the cuvette. not the orginal milk sample. The milk sample was not diluted enough and therefore the concentration of protein in milk sample was higher than last point plotted on the graph. Therefore. the protein concentration was extrapolated instead of interpolated. Most of the students understood the concept of the standard graph and the use of interpolation or extrapolation. DAY 22 INTRODUCTION TO ELECTROPHORESIS Mixes 1. Explain how'amino acids differ from one another. 2. Be able to tell whether the R group of an amino acid is charged and identfy which charge it would have in an aicd solution and which charge it would have in a basic solution. 3. To explain and demonstrate electrophoresis of proteins. Materials 1. Overhead transparency of amino acids showing neutral amino acids. amino acids with extra acid group and amino acid with extra amino group. 2. Overhead transparency of general structure of amino acids in acid and basic solutions. 3. Amino acid problem worksheet. Procedure 1. Review general structure of an amino acid. 2. Identify those amino acids that have an extra acid group, glutamic acid and aspartlc acid. 9. 10. 11. 110 Identify those amino aacids that have an extra amino group. arginine. lysine, and histidine. Review the definitions of acids and bases. Explain why amino acids are positively charged in acid solution and negatively charged in basic solution. Explain the role the extra acid groups in creating a net positive charge in proteins in a basic solution of pH 8.6. Ask students to predict whether a positively charge protein will be attracted to a positive or negative charge. Demonstrate and explain the use of the basic electrophoresis equipment. Demonstrate the production of an agarose gel. Demonstrate the use of Drummond pipettes to fill the wells. Assign the amino acid problem worksheet. and assist students. NoteLandJneedotaLmateriaLs 1. Students worked on the amino acid problem worksheet over a period of three days. Most were able to understand what they were doing. Several students Just did not feel comfortable and failed to complete the assignment. Several others felt it helped them to understand how electrophoresis works. DAY 23 Laboratory Exercise: PROTEIN ELECTROPHORESIS Dbleotlm 1.To separate protein molecules by charge using electrophoresis in agarose gel. W 1. Lab materials Tris-glycine buffer pH 8.6 (2.8 g Glycine and 0.4 g Tris base per liter) Cytochrome c 10 mg/mL in 50% glycerol 111 Myoglobin 10 mgAmL in 50% glycerol Serum albumin 10 mg/mL in 50 % glycerol 0.1% bromophenol blue Micropipettes (Drummond pipette) small beaker or test tube Procedure 1. Agarose gels were prepared ahead of time due to lack of sufficient balances sensitive enough to weigh 2.6 grams of agarose. 2. Explain and demostrate electrophoresis technique. 3. Demonstrate the use of Drummond pipettes. Notee_and_aneodotai_materi.ale 1. The buffer used here is different from buffer used in the lab on the B.R.A.I.N.S. disk. Any mixture of proteins with different isolectric points can be separated by protein electrophoresis. The proteins used here are cytochrome c. myoglobin. and serum albumin. 2. Gels were removed and stained by teacher. DAY 24 Laboratory Exercise: PROTEIN ELECTROPHORESIS. continued theotues 1. To observe and analyze stained electrophoresis gels. . MaterLale Stained gels in zip-lock plastic sandwich bags Protestire 1. Indentify the stained proteins and answer the questions at end of the lab. 2. Complete the Lab Report Outline 3. Discuss results of lab. Notes_and_aneodotai_materlale 1. There were several problems with this first attempt at electrophoresis. Six power sources were used 112 for six groups in second hour and another six groups in forth hour. One of the power sources repeatedly blew fuses. One group forget to connect leads. Other miscellaneous problems resulted in only a few successful gels. However, over all the classes were interested, and the lab was a success. DAY 25 DISCUSSION thflfilllflfl ObJectives listed for Module I, Part 2 Mfllflniilfl 1. Overhead transparencies used during module Procedure 1. Collect Lab Report on electrophoresis and discuss. 2. Return all corrected papers that have not yet been returned. 3. Review all labs and relate to concepts and process skills that have been covered in module. DAY 26 Test (Post test) W DAY 1 CLONING Qaieotluee: 1. Summarize the process of transcription and translation in protein synthesis. 2. Define biotechnology. 3. Define the following terms and use them in context: tissue culture, totipotent. differentiation, clone, and callus. 11mm: 1. Pre-test 2. Overhead transparencies of DNA and protein synthesis. 3. Overhead transparency of plant cloning example- carrots 113 4. Overhead transparency of an animal cloning example- implantation of frog nucleus from intestine cell to embryo. 5. Monsanto Booklets “Of The Earth: Agriculture and the New Biology“ - enough for entire class 6. Vocabulary list with definitions. see Appendix E. Procedure: 1. Administer pre-test. 2. Write the following vocabulary words on the board: biotechnOIOQY. tissue culture, totipotent. differentiation, clone, and callus. Instruct students to write the definitions in their notes as they are covered. 3. Define biotechnology as the use of living organisms to produce products which are technically, econicallly and scientifically useful. Discuss. 4. Review DNA and protein synthesis as necessary. 5. Pass out Monsanto Booklets. These can be numbered and assigned to students, then collected at end of module. Ask students to turn to page 7. Assign reading pages 7 and 8. 6. Define the following terms and use them in context: tissue culture, totipotent. differentiation. clone, and callus. 7. Repeat definitions above until students understand that tissue culture is the technique of growing a whole organism from a single engineered cell or piece of tissue. This is possible because all cells are totipotent. that is, each cell carries all the genetic information it needs to become a whole organism. Most of these genes are turned off during differentiation when cells become specialised. Under the right conditions cells form callus which is undifferentiated again similar to a feritiled egg. 8. If time permits, discuss some applications of tissue culture. Notes_and..ane.c.dotal_materi.al_a: 1. Frequent reference was made to each of the vocabulary words above. Frequent reference must be 114 made to the term totipotent. even though it may seem repetitive. By the end the end of the hour. the students were asking questions and caring on a discussion using the vocabulary words. 2. Student interest was high. They were especially fascinated by the term totipotent. but understanding this term seemed to be key. DAY 2 PREPARATION FOR LAB Oblectlxee: 1. Describe how in some organisms, African Violet, for example. differentiation appears to be reversible and clones can be made by tissue culture. 2. Demonstrate tissue culture lab. Materlale: Monsanto Booklet Laboratory exercise: Effects Of Two Plant Hormones on Culture of African Violet Tissue Materials for Lab see day 3, sufficient for demonstration Procedure: 1. Discuss vocabulary and concepts from previous day. 2. Discuss reading from Monsanto booklet. 3. Demonstrate tissue culture lab. Notee_and_anecdotalumaterlalo: 1. Students were assigned to groups of four. All the steps were demostrated and questions answered. DAY 3 Laboratory Exercise: EFFECTS OF TWO PLANT HORMONES ON CULTURE OF AFRICAN VIOLET TISSUE leectlxee: 1. To recognize that different hormones affect plant cells in different ways to produce various types of growth. 2. To practice sterile technique. 115 3. To develop a practice of keeping accurate, consistent record keeping over long periods of time. MALoLLaLS: See lab for lab materials. Procedure: 1. Follow lab procedures. Notee_and_anecdotal_meterlalo: 1. It was explained that observations would be made at weekly intervals. It was found best not to emphasize effects of different hormones at this time. Students were told what patterns of growth should be seen as weeks progressed, but this would be discussed more later. Results of hormones was not as distinct as in previous year when tobacco leaves were used. The reason is probably that African Violets have smaller leaves with more veins. Cytokinin is carried in the veins and would change results. The African Violets also grow more slowly, but the advantage is that they are readily available. Later in the year two students cultured caulifower, modifying instructions in Amonloon Blolooxglooohon March 1988. Growth appears to be faster than African Violet or Tobacco, however small pieces of cauliflower work best. The plant hormones appeared to produce the expected effect better than with African Violet. DAY 4 GENE REGULATION Doiectlm: 1. Describe the role of RNA polymerase and a promoter in transcription of mRNA. 2. Draw a diagram illustrating the lactose operon (lac operon) and describe how it functions. 3. Describe how lactose induces the production of lactase in the lactose operon, thus “turning on“ that gene. Materials: Diagram of lac operon (from Blologx by Campbell) 116 Overhead transparency of above diagram Vocabulary list with definitions. see Appendix E Procedure: 1. Write the following vocabulary words on the board: RNA polymerase. promoter, structural genes, regulator genes, repressor protein, operator, and operon. Instruct students to write the definitions in their notes as they are covered. Explain the relationship between RNA polymerase and the promoter. RNA polymerase is an enzyme that Joins nucleotides when mRNA is being transcribed, while the promoter is site for binding RNA polymerase so mRNA can be made. Define each of above vocabulary words, referring to overhead transparency of lac operon. Explain what happens when lactose is absent and when lactose is present. For example. when lactose is absent. the regulator gene codes for a repressor protein that regulates the strutural that binds to the operator blocking the promoter. When the RNA plymerase cannot bind to the promoter, the structural genes will not produce mRNA to produce lactase. The system is turned off. Notee_and_anec.dotal_materi_a.l_s: 1. It is very important to establish the function of the RNA polymerase before going further. DAY 5 Laboratory Exercise: EFFECT OF LACTOSE ON LAC OPERON: A STUDY IN GENE REGULATION thflfilllfifiz 1. 2. To introduce students to sterile technique. To inoculate cultures. 11mm: 1. Lab materials E. coli culture Inoculating loop Glucose broth (0.05% sugar) in a test tube lactose broth (0.05% sugar) in a test tube Marker 117 Incubator set at 37°C 2. worksheet “Recombinant DNA“, see Appendix E Procedure: 1. Demonstrate sterile technique of flaming inoculating, etc, used to transfer E; coil from stock culture tube to sterile glucose and lactose tube. Allow 15 to 20 minutes for students to inoculate culture tubes. Remainder of class can be used to explain rest of lab. Stress that O-nitrophenyl-beta-D-galactopyranoside (O-NPG) is an alternative substrate for the enzyme lactase. If lactase is present it will react with lactose goo O-NPG. The O-NPG will be cleaved. Producing a yellow color which is evidence of enzyme action. If time permits, allow students to work on worksheet. Assign remainder for homework. Motes_and_ane.cdotal_materiale: The temptation might be present to inoculate it yourself. However, this short exercise in sterile technique will help prepare students for lab with the plasmid. If only a limited number of stock cultures are available to students, they will have to share. This allows for closer supervision of students with cultures. DAY 6 COMPLETE LAB AND DISCUSSION W: 1. To determine if lactose will induce the production of B-galactosidase (lactase) in E. coli. To detect the presence of the enzyme lactase by its colored end product. mm: 24 hour culture from previous day O-nitrophenyl-beta-D-galactopyranoside (O-NPG) Eye droppers 118 Overhead transparency Computer program “Lac Operon“ Procedure: 1. Ten drops of O-NPG are placed into each tube. The group will observe for yellow color at 10, 20 and 30 minute intervals. 2. Discussion with repeated reference to vocabulary. Continual reminder that lactase enzyme will not be produced unless RNA polymerase can bind to DNA to produce mRNA. 3. While waiting for the color change. a short very simple program called Lac Operon from the Summit Program at Michigan Technilogical University was shown to the class. This program has no graphics but demonstrates the step by step effect of turning on the lactase gene if lactose is present and turning off the gene if lactose is absent. It takes about five to ten minutes to view the simple program. NoteLandJnecdotaLmateriaLa: 1. This not an easy concept to get across. With continual reference to overhead transparency and what is happening in the test tube, the concept can be understood. Remember. keep it simple. 2. Students are not comfortable calling the enzyme B-galactosidase. Because the sugar is called lactose they prefer to enzyme lactase. DAY 7 RECOMBINANT DNA Doieotlxea: 1. Describe the techniques used in recombinant DNA experiments. 2. Describe the action of restriction enzymes, ligases and plasmids in recombinant DNA experiments. Materials: 1. Overhead transparency of recombinant DNA steps 2. Vocabulary list with definitions, see Appendix E 2. Worksheet - restriction enzyme acitivity 119 Procedure: 1. Explain that recombinant DNA is a highly publicized and controversial technique in which DNA from two or more species is Joined. 2. Explain the role of restriction enzymes, ligases and plasmids. When the same restriction enzyme cleaves DNA from two different species. the resulting DNA segments have complementary ends (sticky ends). If the segments of the two species are then mixed and DNA ligase is added the segments Join. This recombinant DNA can then be inserted into bacterial cells by incorporating it into plasmids. small circular strands of DNA which replicate independently of the bacterial chromosome. 3. Once the basic technique of recombinant DNA is understood, use the Monsanto booklet to discuss applications. Notes_and_anecdotal_materlels: 1. The worksheet on restriction enzyme activity may be too difficult to cover the first day. Discussion may need to continue on Day 9 before discussing the lab. DAY 8 VCR “LIFE PATENT PENDING“ W3 1. Introduce recombinant DNA and the concept of patenting its products. 2. Summarize the ethical and other obJections that have been raised against recombinant DNA studies and give potential practical and research applications. Materials: Nova Program :Life Patent Pending“ 120 Noteo_and_anecdotal_materials: 1. This program introduces recombinant DNA and some applications. It is one of the earlier films and it discusses the criticism of recombinant DNA research. Adversaries accuse scientists of playing God, of endangering public safety by inventing toxic new organisms, and of slowing free flow of scientific information because of possible patent on recombinant products. It also discusses the regulations on the industry. DAY 9 PREPARATION FOR LAB Doleetlxee: 1. Transform the bacterium Eoohorlohio ooli from ampicillin sensitive to ampicillin resistant with the plasmid pBR322. Materials: Sample lab materials Procedure: 1. Demonstrate or explain each step of two day lab. Notee_and_anecdotal_materlalo: 1. This is a good time to discuss the reason for each step. DAY 10 Laboratory Exercise: TRANSFORAMTION OF THE BACTERIA ESQHERLQHLA,§QL1 with pBR322 PLASMID ObJectlm: 1. To transform the bacterium Eoohonlohlo ooll from ampicillin sensitive to ampicillin resistant with the plasmid pBR322. Materials: 1. See lab for lab materials. Procedure: 1. Follow lab procedure. Stop at step 8, after heat shock the tubes can kept on ice overnight. 121 W: 1. The more organized the class and materials, the easier this lab will go. Make sure everything is properly labeled. DAY 11 Laboratory Exercise. continued Dblectlxeo: 1. To transform the bacterium Boohoolonlo ooll from ampicillin sensitive to ampicillin resistant with the plasmid pBR322. Materials: see previous day E£Q££§M£13 Follow procedure from step 9. Notee_and_anecdotal_materlale: 1. The students tried very hard to follow the exacting procedures. Their technique was impressive for sophomores. DAY 12 VCR GEOMETRY OF LIFE QDIoELLloS: 1. Observe growth of ampicillim sensitive and ampicillin resistant bacteria. 2. Summarize the ethical and other obJections that have been raised against recombinant DNA studies and give potential practical and research applications. Materials: Video tape “Geometry of Life" Procedure: 1. Allow a few minutes at the beginning of the hour to observe plates and breifly discuss. 2. Show video tape. 122 NoteLandJnecdotaLmaterLals: 1. This is a relatively new film. The graphics are well done. There is discussion of gene regulation. electrophoresis of DNA. and recombinant DNA. Day 13 VCR Monsanto leectluea: 1. Give potential practical and research applications of recombinant DNA. Materials: 1. Video tape “Of The Earth: Agriculture and the New Biology“ 28 minutes. 2. Video tape “BST: Continuing a Dairy Tradition“ 11 minutes Procedure: 1. Show the films, and discuss. Notee_.and_ane.cdot.al_matcrl.alo: 1. The first film complements the booklet of the same name. It includes animation describing recombinant technology and how plants are engineered. Available on free-loan basis from Venard Films Ltd. P.O. Box 1332, Peoria, IL 61654, phone: (309) 699-3911. (Released 1986) 2. The second film gives a general overview of the nature of the protein, its use and its safety. Available from same address as above. DAY 14 VCR EDITED SEGMENTS WITH DISCUSSION ObJectives: 1. Give potential practical and research applications of recombinant DNA. Materielo: 1. Edited segments of various video tapes that show tissue culture or recombinant DNA, some of the segments have already been as part of other video tapes. 123 Procedure: 1. Show a segment of the video tape, turn off the tape and discuss. Repeat through tape. Notewdmiecdotaunaterlale: 1. Students appear to like this format. It allows them to ask questions about what is being shown on the tape. DAY 15 REVIEW QbJecthee: ObJectives for Module II. Materials- I'Of the Earth: Agriculture and the New Biology, Monsanto. 1986. Entire Booklet. Pages 1 - 24. Overhead transparencies Vocabulary list with definitions, see Appendix E Procedure: 1. Review each concept- tissue culture, gene expression and recombinant DNA. For each concept, review vocabulary, labs and applications. NoteeJndJneodotaLmateriaie: 1. The Monsanto booklet was used again during the review. DAY 16 TEST APPENDIX D PRE-TESTS AND POST-TESTS APPENDIX D PRE-TESTS AND POST-TESTS Module I was taught in two parts, Inorganic Chemistry and Organic Chemistry for beginning biology students. Each part had its own pre-test and post-test. Module II, Biotechnology, was also monitored by a pre-test and post-test. The three sets of pre-post tests are included in this appendix. The statistical analysis of the pre-post tests was discussed in Chapter 4 Evaluation. Each pre-test and post-test consisted of 8 questions. For grading purposes, a longer unit test was adninistered the same day as each of the post-tests. The questions analyzed as the post-test were the first 8 questions of the unit test. The three unit tests are also included in this appendix. The means for the three unit tests were 89.2%, 88.0%, and 83.3% respectively. 124 2. 125 INTRODUCTORY CHEMISTRY PRE-TEST AND POST-TEST The diagram represents a(n) A. compound 8. ion C. atom D. molecule Give the reason for your answer to question 1. Which atom, sodium (A) or chlorine (B) would gain an electron if the two atoms combine with each other? /o‘-E;\- «:88 - \\ . / e-e\ / . ' C73 96? so? e€\\\@//ee Give the reason for your answer to question 3. Sodium chloride. an ionic coupound, is dissolved In water. The solution contains _ A. sodium chloride molecules and water molecules 8. sodium ions. chloride ions, and water molecules C. sodium chloride molecules and hydroxide ions D. sodium ions. chloride ions, and hydroxide ions Give the reason for your answer to question 5. A solution with a pH of 5 has A. greater concentration of hydrogen ions than hydroxide ions 8. greater concentration of hydroxide ions than hydrogen ions C. equal concentration of hydrogen and hydroxide ions D. greater concentration of hydrogen ions than a solution with a pH of 4. Give the reason for your answer to question 7. 126 ORGANIC CHHISTRY PRE-TEST 1. much of the following is an organic compound? 2. Give the reason for your answer to question 1. 3. A conpound contains two carbon atone. six hydrogen atone. and one oxygen atom. Imich of the following is a corrrect structural formula for a molecule of this compound? 1: I . n 0 H H . H n A. I B. I I C- I D: I I u—c—u—x—n—u—c u—c—c—n—n C—N—H—N—"-° "_¢-c-'°"" I | I I I w H-C :n .u 4. Give the reason for your answer to question 3. Use the following diagram to answer questions 5—8. H I H—C—O—H o i I II o H-C-O—C-R "\ /,', \ /" H H o o c 9\ I I H u a . H—N—C—C—O—H H—C—O-C—n o/ I o I / o H‘ - \ H—C—H o H . I I H 1 II C C H H—C—O-C—R I I I H H ‘1’ A 8 C H 5. Which of the molecules is a building block of protein? 6. Which of the molecules is a lipid? 7. Which of the molecules is a building block of carbohydrate? 8. Which molecule is produced during photosynthesis? 127' ORGANIC CHEMISTRY POST-TEST 1. Which of the following is an organic compound? II. 820 a. xcz. c. 11250,, 0. c3118 2. A compound contains two carbon atou~. six hydrogen atoms, and one oxygen atom. Which of the following is a corrrect structural formula for a molecule of this compound? II I . n 0 H H H n A I B I ; C- I '°- I I H—C—H—N—H—N—C H—C—C—n—H C—u—H—H—H—O N—C—C—O—‘u l E I I I O u H—C on .x 3. When using the spectrophotometer to measure the amount of starch-iodine mixture in a test tube, which statement is true? A. the 8 transmittance decreases as the concentration of starch decreases B. the % transmittance increases as the concentration of starch decreases C. the absorbance increases as the concentration of starch decreases 4. Electrophoresis can be used to separate proteins. The most important is the A. size of the protein 8. charge of the protein C. number of neutral amino aci¢§ Use the following diagrams to answer questions 5- 8. l H—C-O—H °\ H I :1 _fl \ /H \ I. C H H o i c \ I I H. II H // H—N—C-C—O—H H—C-O-C-fl o/ I o H-c-H : o .. . I I/ \H I H H _ ? A ' B C H . Which of the molecules Is a building block of protein? 5 6. Which of the molecules is a lipid? 7. Which of the molecules is a monosaccharide? 8 . Which molecule contains an amino group? i. 128 Biotechnology Pro-test Althoud: hemoglobin is not found in muscle cells. it is very abundant in red blood cells. Why? A. B. A. B. D. 8. The hemoglobin gene is present in red blood cells but not In Imiscle cells. The hemoglobin gene is present in both cell types but is only active in red blood cells but not in muscle cells. Muscle cells only have one copy of the hemoglobin gene while red blood cells have many. Hemoglobin is prodiced in both cell types but is rapidly destroyed in mscle cells. With recoobInant NA technology we are currently capable of allowing organises to make new proteins creating entirely new organisms both a and b The technology is available to grow a whole plant from a piece of tissue or a gene engineered cell. True False The direction of transfer of genetic information in most living things is protein > DNA > mRNA DNA ) mRNA > protein DNA > tRNA > protein RNA > DNA ) mRNA > protein Explain the reason for your answer to question 4. A plasmid is A fragment of a bacterial chromosome A circle of 111A separate from a bacterial chromosome A virus that infects a bacteria Suppose a pharmaceutical cmpany planned to clone the gene for human insulin. Which of the following steps would not be involved in the process? Joining ends of DNA by an enzyme Breaking human DNA by an enzyme breaking human DNA into indivimal nucleotides Joining human DNA to a plasnid Explain the reason for your answer to question 7. 129 Biotechnology Post-test The external appearance of trait usually depends upon: type of protein produced types of sugars produced rate of protein synthesis creating an enzyme that catalyzes the reverse reaction . The sugar lactose induces synthesis of the enzyme lactase. An E. coli (bacteria) cell is presented for the first time with the sugar lactose as a potential food source. Which of the following occurs when the lactose enters the cell? A. Repressor protein attaches to the regulator B. Lactose binds to the repressor protein C. Lactose binds to the regulator D. The repressor protein binds to RNA polymerase 3. Althoug: hemoglobin is not found in muscle cells, it is very abundant in red blood cells. Why? A. The hemoglobin gene is present in red blood cells but not in muscle cells. B. The hemoglobin gene is present In both cell types but Is only active in red blood cells but not in muscle cells. C. Muscle cells only have one copy of the hemoglobin gene while red blood cells have many. D. Hemoglobin is prodiced in both cell types but is rapidly destroyed in nuscle cells. Each cell carries all the genetic information it needs to become a whole organise. This is called callus B. clone C. differention D. totipotent The direction of transfer of genetic information in most living things is protein ) DNA ) mRNA 8. DNA > mRNA ) protein DNA > tRNA > protein D. RNA > DNA > mRNA > protein A plamid is A fragrant of a bacterial chromosome A circle of DNA separate fun a bacterial chrmosome A virus that infects a bacteria Suppou a pharmaceutical company planned to clone the gene for human insulin. Which of the following steps would not, be involved in the process? Joining ends of DNA by an enzyme Breaking human WA by an enzyme breaking human MA into individual nucleotides Joining human DNA to a plasnid . In recoubinant DNA technology. DNA is cut by . restriction enzymes B.plasnids C. ligases D. polymerase )0 CPU) 2. 3. 130 INTRODUCTORY CHEMISTRY UNIT TEST The diagram represents a(n) A. compound 8. ion C. atom D. molecule Give the reason for your answer to question 1. Which atom, sodium (A) or chlorine (B) would gain an electron if the two atoms combine with each other? see/j) B Give the reason for your answer to question 3. Sodium chloride. an ionic compound, is dissolved in water. The solution contains A. sodium chloride molecules and water molecules 8. sodium Ions, chloride ions, and water molecules C. sodium chloride molecules and hydroxide ions 0. sodium Ions, chloride ions, and hydroxide ions lee the reason for your answer to question 5. A solution with a pH of 5 has A. greater concentration of hydrogen ions than hydroxide ions 8. greater concentration of hydroxide ions than hydrogen ions 0. equal concentration of hydrogen and hydroxide ions D. greater concentration of hydrogen ions than a solution with a pH of 4. Give the reason for your answer to question 7. 131 9. In a covalent bond. electrons are A. transferred 3. gained C. shared D. lost 10. In an uncablned atom. the nuaber of protons must equal the nuwer of A. electrons D. shells C. neutrons D. charges 11. A solution which contains a greater concentration of hydroxide ions than hydrogen ion is an DNA ) mRNA B. INA ) mRNA > protein lllA > tRNA > protein D. RNA > DNA > mRNA > protein A plauid is A fragnent of a bacterial chromosome A circle of DNA separate from a bacterial chromosome A virus that infects a bacteria «1 OU)O‘ 0) 0| 3 Suppose a pharmaceutical company planned to clone the gene for human insulin. lfllich of the following steps would not be involved in the process? Joining ends of DNA by an enzyme Breaking human DNA by an enzyme breaking human DNA into individial nucleotides Joining human INA to a plamid P°"? 8. In recalbinant M technology. DNA is cut by A. restriction enzymes B.plasnids C. ligases D. polymerase 140 TRUE/FALSE 9. 10' 11. 12. 13. 14. flat A. C. D. E. E. 6. He I. 15. 16. 17. 18. 19. 20. 21. Chains of amino acids make a protein. The whole DNA strand of a chromsome is thouglt to be active in coding one protein. The whole DNA strand of a chromosome is thought to be active in every cell. The DNA controls the cell by controlling all the protein made in the cell. A gene is that portion of the DNA that codes for one protein. The process of practicing mRNA from DNA is called transcription. ching I clone biotechnology callus totipotent differientiation promoter RNA polymerase tissue culture electrophoresis The use of living organisms to produce products which are technically, econicallly and scientifically useful. The technique of growing a whole organimn from a single engineered cell or piece of tissue. An exact copy of a gene, a cell, a bacteria, etc. Undifferentiated or unorganized tissue which grows from a plant cell or piece of leaf when it is placed on media containing special hormones. Cells specialized into certain tissues and organs. An enzyme that Joins nucleotides when mRNA is being transcribed Site for binding RNA polymerase so mRNA can be made . Movement of charged molecules under the influence of an electric field 141 MULTIPLE CHOICE 23. One type of specialized cell (a leaf) differs from another (a root) in the same plant because A. they have made different enzymes 3. they have different DNA C. both A and B D. neither A and B 24. When plasmids are Joined with foreign DNA. the enzyme that connects the DNA is called a A. restriction enzyme 8. polymerase C. ligase D. peptidase 25. Which of the following do not Join together easily? EcoRi and Hind III are restriction enzwmes. A. human DNA cut with EcoR1--chlmpanzee DNA cut with EcoRi 8. procaryotic DNA cut woth Nind III--eucaryotic DNA cut with Nlnd III C. mouse spleen DNA cut with EcoR1--mouse kidney DNA cut with EcoRl D. E. coli DNA cut with EcoR1--mouse DNA cuth with Hind III E. mouse DNA cut with liind III--elephant DNA cut with ilind 26. Which letter or letters indicates the location of a gene or genes that code for the enyzme lactase? 27. Which letter indicates the binding site for the enzyme RNA polymerase? 28. Which letter indicates the binding site for the repressor protein? 29. 30. 31. (No 142 Which letter indicates the gene that codes for the repressor protein? Which letter indicates the repressor protein? The promoter A. codes for the repressor proteins 8. is the binding site for the repressor protein C. is the binding site for RNA polymerase D. is the binding site for lactose sugar suppose a gene has the DNA nucleotide sequence 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 C T C G C A T C C T T C G G A A A real gene could be this alert. but for our purposes this will suffice.) Which one of the following mtations would probably produce the greatest change in the activity of the protein for which this gene codes? . substitution of A for G in position 3 . deletion of the C at position 5 . deletion of the A at position 16 addition of a 6 between position 14 and 15 POW, Restriction enzymes are useful in reconbinant NA technique because they * A. cut NA at specific sites B. restrict the nunber of nucleotides that can be removed at one time C. restore the bonds in the NA backbone D. make NA from mRNA A genetic engineer prepares DNA fragnents from two species and mixes them together. Two of the many fragnents are shown below. Iniich one of the following statements is correct? TTCC ATGC A. sticky ends were produced 8. The two fragnents shown above will Join by complementary base pairing C. The two fragnents were prepared by two different restriction enzymes D. A single restriction enzyme was used to cut at different locations in the two types of DNA I43 35. If you were to arrange the following steps in gene cloning in order. which step would be third? A The plasmid is inserted into the bacterial host cell 8. The plasmid is cut by a restriction enzyme ’ C. DNA molecules coding for the desired product are isolated D. Restriction enzymes are used to cut DNA. producing sticky ends E. The plasmid DNA and the cloned gene are Joined and sealed by ligase Use the following information to answer questions 36-39. Use True-False. Normel bacteria growing on ampicillin would be killed. If the bacteria are treated with calcium chloride and a plamnid containing a gene for ampicillin resistance is inserted into these bacteria. the following results are obtained. ( + indicates presence of plasmid: - indicates absence of plasmid). I: + Is 5 a g) CONTROL ANPICILLIN 36. The bacteria containing the plasmid will not grow on the ampicillin. 37. The bacteria not containing the plasmid will grow on the ampicillin. 38. The calcium chloride makes the cell membranes more permeable so the palsmid will enter. 39. The gene for ampicillin resistance allows the bacteria to produce an enzyme that destroys ampicillin. 144 Essay 1. Assume that you are working for a large pharmaceutical conpany in their research division. Your goal is to produce interferon in large quantity by genetic engineering techniques. Ybu are provided with a DNA fragment, containing the interferon gene, which had been isolated using restriction enzyme A. YOu know that the interferon gene is flanked on both ends by recognition sites for 8. another restriction enzmme. An E. coil plasmid with single 8 recognition site is available. What would you do? Describe all the steps of your experiemnt. 2. Explain the reason for your answer to question 23. 3. Explain how tissue culture could be used with recombinant DNA. 4. State two uses of recombinant DNA that you think are good uses of this technology. State why you think the use the recombinant DNA in your two examples would be beneficial to the world. APPENDIX E SUPPLEMENTARY HANDOUTS 145 APPENDIX E SUPPLEMENTARY HANDOUTS CHEM I CAL MODEL BU I LDI NG Cabins the following stoms to form molecules. Draw a possible structural formula. 1.1C+4l-l 2. 2C+ 6H 3.ZC+4II 4.1C+2!i+10 5.1C+2N+20 6. 2C+6Il+10 7.3C+8ll+30 8.2C+5Il+20+1N 9.C+12ii+60 (hint: both 0 on sane C) (Nany conbinations Possible) 146 INTROWCTORY CNDIISTRY WORKSI'IEE'I' 1. refers to an atom that has lost or gained electrons. 2. llany substances come apart. or . into ions when whey dissolve in water. 3. A solution of pli _ has equal concentrations of 11+ and Oil- ions. 4. A solution of pll 5 is __(acid or base). 5. Solutions with p11 values belwo 7 are called __ and have __l-l+ (more or less) than solutions with pH values above 7. 6. A base . or alkali. is a substance that releases in water. 7. A substance in which the ll+ ions of an acid have replaced by other positively charged ions is a . 8. A sibstance that maintains a fairly constant pii is a 9. A bond is formed by atoms during electrons. 10. An bond is formed by atans giving up or accepting electrons. 147 ENZYME STRUCTURE AND FUNCTION 1. What are the building blocks of a protein? 2. Why is the nunber of different proteins virtually unlimited? 3. What is the primary structure of a protein? 4. What is meant by conformation of a protein? 5. Identify at least one factor that prodices the three dimensional structure of a protein. 6. What is an enzyme? 7. What is meant by the active site of an enzyme? 8. What happens when a protein is denatured? 9. What are some factors that can denature a protein? 10. What happens to the function of an enzyme when it is denatured? I48 AHINO ACID PROBLEHS Describe each amino acid by filling in the appropriate blanks. NAHE CHEMICAL PQRHULA SULFUR ACIDIC CHARGE CHARGE PRESENT? BASIC ON R ON R OR GROUP GROUP NEUTRAL PH 7 pH 8.6 1. Lysine 2. H H O l I ll H— N -C -C -OH I H-C-H I S-H 2. Glutuic acid 3. H N O I I II N- N -C -C -ON I N-C-N I 080 I OH 4. Arginine 5. Hethionine 6. Prollne 10. 11. 12. 13. 14. 15. 16. 17. 149 BIOTECHNOLOGY UNIT Biotechnology - the use of living organises to produce proacts which are technically. econicallly and scientifically useful. gene- that portion of NA molecule that codes for one protein nucleotide base - one of four bases. (A.T.G.C) protein- molecule comosed of amino acids Tissue culture — the technique of growing a whole organimn from a single engineered cell or piece of tissue. Clone- an exact copy of a gene. a cell. a bacteria. etc. sterile technique- without contamination or infection Callus- undifferentiated or unorganized tissue which grows from a plant cell or piece of leaf when it is placed on media containing special hormones. Totipotent- each cell carries all the genetic information it needs to become a whole organim. Differentiation- to become specialised: cells specialized into certain tissues and organs. Gene regilation- turning off or on genes RNA polymerase- an enzyme that Joins nucleotides when mRNA is being transcribed Promoter- site for binding RNA polymerase so mRNA can be made Structural genes- those genes that code for the proteins needed by the cell. either enzymes or proteins that have structural function. Regilator gene- gene that codes for a repressor protein that regulates the strutural genes. Operator- site where repressor protein binds blocking the promoter lac operon- group of genes including the promoter, operator and structural genes that control production of lactase 10. 19. 150 electrophoresis- movement of charged molecules under the influence of an electric field NA fingerprint- identifcation of individuals using electrophoresis of NA segments genetic engineering- also called reconbinant DNA reconbinant NA- the technique of isolating DNA molecules and inserting them into the NA of a cell- 'reconbining DNA' rNA technology- recolbinant NA technology gene aliclng- also called reconbinant NA or genetic engineering plamnids- mall circular strands of NA which replicate independently of the bacterial chromosome. vector- the messenger which carries new genes into cells. Plamnids currently are the preferred vectors. but viruses may be used as vectors also. restriction enzymes- enzymes that can I'cut" a gene out of a piece of NA. ligase- an enzyme that splices segaents of NA together. 'sticky ends'- single-stranded cowlementary ends of NA that stick to each other by hydrogen bonding. transformation- change from one form to another brought about by transfer of NA. 151 RflflRKHHNINDMEACHVHY Directions: 1. Select one of the DNA sequences below. 2. Select one of the restriction enzymes. At any recognition site for that enzyme place an '/' to indicate the site of cleavage. 3. This may be repeated with other restriction enzymes. You may want to use a different color for each restriction enzyme. 1) CCAGTCGTTAACGAATTCGTCGACGTCGAC GGTCAGCAATTGCTTAAGCAGCTGCAGCTG 2) ACGGGTTAACCCAATGGATCCCAAGTTAACGGTAC TGCCCAATTGGGTTACCTAGGGTTCAATTGCCATG Restriction enzyme* Recognition Site** Eco RI G/AATTC Sam 31 G/GATCC Hpa I GTT/AAC Sal I _ G/TCGAC * Each enzyme recognizes a symmetrical sequence ** The / indicates the site of cleavage 152 Recoflinant NA 1. How did farmers inprove crops before recombinant DNA technology (also called genetic engineering) was available? that is the advantage of using genetic engineering to agriculture? ‘ Describe the function of each of following in recombinant NA: a) plamids b) restrict ion enzymes c) ligase enzymes ° Explain what is meant by 'sticky ends'? How is tissue culture inortant in recmbinant NA? Briefly discuss each application of reconbinant DNA: a) Insect resistance b) herbicide tolerance c) virus resistance in plants d) resistance to frost damage e) nitrogen fixation f) tissue plamainogen activator (tPA) g) atrial peptides h) bovine somatotropin (BST) i) humulln (insulin) B I BL I OGRAPI'IY BIBLIOGRAPHY Bohnsack. C.W. (1989). Cytokinin induced cell division and differentiation using intact plants. The Weber. 51(2). 106. Dixon. L. (1988). Teaching recombinant DNA Technology in high school biology courses. The_AmenTeeh W. 50(6). 368-373. Gardner. A.N. (1988). Biotechnology in 3 days. The AmerIeen_BIQIegx_Teeeber. 50(7). 446. Gattozzi. L.N.. Hagstrom. E.F.. Rediess. M.E.. and Salminen. N.D. (1988). Bargain-basement electrophoresis. The_5eTehee_Teeehe;. 55(7). 22-25. Haldmman. J.H.. and Ellis. J.P. (1988). Using cauliflower to demonstrate plant tissue culture. TbLAmerieeLBIeiegLJeaeber. 50(3). 154-159. Igelsrud. D.. and Leonard. W.H. (1988). 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