BISC 219/2009:Creating a Transgenic Organism
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 Set-up of the tranformation |
| Independently | 9/11 - 9/17 | transfer of explants to selective medium; induce shoots br> Selecting for transformants |
| 4 | 9/29 - 10/5 | transfer shoots to root inducing medium Transfer to rooting medium |
| 7 | 10/26 - 10/30 | transfer plantlets to soil Tranfering the Plantlets to soil |
| 8 | 11/4 - 11/10 | single leaf-disc PCR Protocols for Structural Evidence for Transgenic Plants |
| 9 | 11/11 - 11/17 | Restriction enzyme digest of PCR reactions Agarose gel electrophoresis of digested PCR |
| 10 | 11/18 - 11/24 | Spectrophotometric GUS enzyme activity assay Leaf Extract Preparation Spectrophotometric Assay for GUS activity |
| 11 | 11/30 - 12/4 | Data Analysis Workshop Instructions for Analyzing the data on the putative transgenic and control plants |
| Dec 11 | 12/11 | Plant Genetic Engineering Paper Due for all students |
Experimental Objectives:
In this semester long experiment you will:
- Master aspectic technique and tissue culture;
- 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;
- using PCR to test directly for the presence of the introduced genes.
| Concepts | Genes/Organisms | Techniques/Skills |
|---|---|---|
| Effect of random gene insertion in a large genome | Vector bacterium: Agrobacterium tumefaciens | Aseptic tissue culture |
| Plant totipotency | Transgenic recipient: Tobacco plant- Nicotiana tobacum | DNA Extraction |
| Regulation of gene expression | Transgene: gusA gene from Escherichia coli encoding beta-glucoronidase | 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
Transgenic Plants: Media Recipes
Lab 1: Creating the Transgenic Plant: Day 1
Outside of Lab in the first week: Creating the Transgenic Plant: Days 3-5
In Lab 4: Creating the Transgenic Plant: Week 4-5
In Lab 7-8: Creating the Transgenic Plant: Week 8
Lab 8:GUS by histochemistry
Lab 8:Structural Evidence for Transgenic Plants: DNA extraction, PCR
Lab 9: RE digestion, Agarose gel electrophoresis of the PCR product
Lab 10:Leaf Extract Preparation for GUS Spectrophotometric Assay
Lab 10:Spectrophotometric Assay for GUS activity
Lab 10:Calculations
Lab 11: Analyzing the all the data on the putative transgenic and control plants
