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GENA Program
A.M. DiLorenzo
September 12, 2008
GENA Teaching Plan
2. What can you tell from this picture? 3. What can you NOT tell from this picture? EXPLORE:
Pre-Lab Questions

1. How does gel electrophoresis separate DNA molecules in mixture? 2. What are the steps of bacterial transformation? What conditions are needed? 3. What are competent cells? 4. How is a plasmid engineered? How is antibiotic resistance transferred between bacterial cells? 5. How do restriction endonucleases function? 6. What are restriction enzymes used in bioengineering? GENA Program
A.M. DiLorenzo
September 12, 2008
Laboratory 6: Molecular Biology
In this lab you will investigate some basic principles of genetic engineering. Plasmids containing specific fragments of foreign DNA will be used to transform E. coli cells, conferring antibiotic (ampicillin) resistance and the Lac + phenotype (ability to metabolize lactose). Restriction enzyme digests of phage lambda DNA will also be used to demonstrate techniques for separating and identifying DNA fragments using gel electrophoresis. Objectives
• Discuss the principles of bacterial transformation • Describe how to prepare competent E. coli cells • Discuss the mechanisms of gene transfer using plasmid vectors • Discuss the transfer of antibiotic resistance genes and tell how to select positively for transformed cells that are • Discuss the mechanisms of action for restriction endonucleases • Discuss how a plasmid can be engineered to include a piece of foreign DNA that alters the phenotype of the • Understand and be able to explain the principles of electrophoresis as they pertain to separating and identifying Bacterial Transformation-Ampicillin resistance: In this exercise, we will introduce competent E. Coli cells to take up the plasmid pAMP which contains a gene for ampicllin resistance. Normally, E. Coli cells are destroyed by the antibiotic ampicillin, but E. Coli cells that have been transformed will be able to grow on agar plates containing ampicillin. Thus, we can select for transformants; those cells that are not transformed will be killed by ampicillin; those that have been transformed will survive. Restriction Enzyme Cleavage of DNA: Restriction endonuclease recognizes specific DNA sequences in double-stranded DNA and digests the DNA at these sites. The result is the production of fragments of DNA of various lengths corresponding to the distance between identical DNA sequences within the chromosome. By taking DNA fragments and systematically reinserting the fragments into an organism with minimal genetic material, it is possible to determine the function of particular gene sequences Electrophoresis: fragments of DNA can be separated by gel electrophoresis when any molecule enters the electrical field, the mobility or speed at which it will move is influenced by the charge (negative charges travel to positive/top pole of gel), the density of the molecule, (the smaller the molecule, the faster it travels), the strength of the electrical field, and the density of the medium (gel) which it is migrating. GENA Program
A.M. DiLorenzo
September 12, 2008
Formative Assessments
1. After growth on ampicillin to select bacteria transformed with a mixture of recombinant DNA containing plasmids, you must identify a clone containing a specific gene sequence. You would: A. Blot transfer clones to membranes, and screen using a radioactive probe complementary to the gene. B. Re-grow bacteria in ampicillin where only transformants containing the gene of interest can grow. C. Culture bacteria in both ampicillin and tetracycline to select for bacteria containing the gene of interest. D. Digest DNA from the plasmid to isolate the gene fragment. E. Do a restriction map of plasmid DNA to identify the correct clone 2. How are restriction enzymes and plasmids used to transform bacteria with a gene for antibiotic resistance? What are the biological processes of transformation in bacteria? 3. What properties of gel electrophoresis allow for separation of DNA fragments? How do you determine DNA fragment GENA Program
A.M. DiLorenzo
September 12, 2008
Study and Review Questions
1. Define biotechnology. 2. What is meant by the term “recombinant DNA technology”? 3. Why are bacteria used as vectors for recombinant DNA technology? 4. What is a plasmid? 5. What do restriction enzymes do and how are they used? 6. Briefly describe the steps involved in cloning a gene. 7. How can transformed bacteria (which carry the gene of interest) be identified and isolated from the non-transformed 8. Describe the process of PCR. 9. Why is PCR an important technique? 10. What are some of the applications of recombinant DNA technology? Address at least one agricultural and one Summative Assessment
2007 AP Biology Free Response Question – see attached page. Final Report of GENA Teaching Plan:
Molecular Biology Lab
Dept of Biology and Molecular Biology Montclair State University Montclair, NJ Jody Dolce Caldwell High School Caldwell, NJ Overview: The GENA teaching plan was designed to introduce upper level high school students to the basic principles of genetic engineering. It involved insertion of plasmids with antibiotic ( ampicillin ) resistance and Lac+ phenotype( ability to metabolize lactose) into E. coli cells. A second part of the Unit included a demonstration of restriction enzyme digest of phage lambda to visualize separation of DNA fragments via electrophoresis. This was performed at both the high school level and in the genetics lab at college level. This allowed a more meaningful dialogue between the college professor and the high school teacher. Evaluation/ Suggestions for improvement: 1. It was noted that for both levels of students it was too ambitious to combine the two components of this lab. This should be separated into a genetic engineering lab and a lab on restriction enzymes, not necessarily combined . 2. Students must be prepared more in basic microbiology before they can appreciate the plasmid transfer. There is need for a basic micro lab before attempting the plasmid lab. 3. Electrophoresis must be connected in student minds to practical use, ie. Forensics, CSI on TV. 4 The protocol for both of these labs must be improved. The vocabulary used might not be familiar to students and add to their confusion. The timing of these topics must follow sufficient exposure to both microbiology and DNA history. 5. A hands-on model of bacterial transformation or a Web site with good animation must be used before the actual lab plasmid transfer. Insufficient visualization at present. 6. Better photos of electrophoresis must be found on line or in books before any possible labs are attempted using restriction enzymes. The photo used to engage students in this lab was too complicated. A simpler photo of electrophoresis should include only a DNA ladder for reference and use of just one enzyme. Once the students can see and understand the value of the restriction enzyme, they will understand the concept of the use of multiple enzymes. 7. Bacterial transformation, restriction enzyme cleavage, and electrophoresis, must each be clearly discussed and understood before the teacher, at any level, should attempt to connect the three. This joint project resulted in slowing down the high school student introduction to these topics. More time had to be spent to be sure of sufficient concept comprehension. Interestingly, at the college level the same need for more time was shown. It is futile to rush through labs if students are mere robots and do not know “why “ they are performing an experiment. As a result of the joint examination of these labs, both high school and college teachers will change their approach in the next academic year. 8. The language/ vocabulary used in the pre-lab, protocol, and post-lab questions must be clarified. Both teachers found that many students are first generation Americans, thus English is not spoken at home. One cannot assume reading comprehension to be of the highest level. 9. Although the GENA Project had as a goal the improvement of high school genetics concepts, a valuable outcome, will be an improvement of labs and teaching at BOTH high school and college levels.


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