BISC 219/2009:Creating a Transgenic Organism
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At the end of this semester long series you will have mastered the following skills and concepts using both microorganisms and a multicellular organism.
Schedule of Experiments
Lab # | Dates | Activity |
---|---|---|
1 | 9/8 - 9/14 | set-up of plant transformation |
Independently | 9/11 - 9/17 | transfer of explants to selective medium; induce shoots |
5 | 10/6 - 10/9 | transfer shoots to root inducing medium |
7 | 10/26 - 10/30 | transfer plantlets to soil |
8 | 11/9 - 11/13 | single leaf-disc PCR |
9 | 11/16 - 11/20 | GUS activity assays; run gel of PCR products |
10 | 11/30 - 12/4 | Data Analysis Workshop |
12/11 | Plant Lab Report Due for all students |
Experimental Objectives:
In this semester long experiment you will:
- use the Agrobacterium system to introduce a foreign gene, the β -glucuronidase gene (gusA) of E. coli, into the cells of tobacco
- take advantage of the totipotency of plant cells and use these transformed cells to regenerate genetically engineered plants
- confirm that the regenerated plants are transformed by
- measuring the activity of the introduced enzyme, β–glucuronidase (GUS) spectrophotometrically and histochemically
- PCR to test directly for the presence of the introduced genes and
- become adept at sterile technique.
Concepts | Genes/Organisms | Techniques/Skills |
---|---|---|
Effect of random gene insertion in a large genome | Bacterial pathogen: Agrobacterium tumefaciens | Aseptic tissue culture |
Plant totipotency | Tobacco plant: Nicotiana tobacum | DNA Extraction |
Regulation of gene expression | gusA gene from E. coli | PCR |
Genetic Engineering and creation of genetically modified organism | Agarose gel electrophoresis | |
Phenotypic Selection | Transfection & selection by antibiotic resistance;
Enzyme function assays: colorimetric & histochemical |
Background
Experiment 1: Media Recipes
Experiment 1: Creating the Transgenic Plant: Day 1
Experiment 1: Creating the Transgenic Plant: Days 3-5
Experiment 1: Creating the Transgenic Plant: Week 4-5
Experiment 1: Creating the Transgenic Plant: Week 8
Experiment 2: Assaying the Transgenic Plants
Experiment 3: Agarose gel electrophoresis of the PCR product
Analyzing the data on the putative transgenic and control plants
Scientific Paper on Plant Genetic Engineering