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
Independently	9/11 - 9/17	transfer of explants to selective medium; induce shoots
4	9/29 - 10/5	transfer shoots to root inducing medium
7	10/26 - 10/30	transfer plantlets to soil
8	11/4 - 11/10	single leaf-disc PCR Protocols for Structural Evidence for Transgenic Plants Histochemical GUS enzyme activity assay Protocols for GUS Activity Assay by Histochemistry
9	11/11 - 11/17	Restriction enzyme digest of PCR reactions Agarose gel electrophoresis of digested PCR Protocols for RE digestion, Agarose gel electrophoresis of the PCR product
10	11/18 - 11/24	Spectrophotometric GUS enzyme activity assay Protocols for Spectrophotometric GUS activity assay Phenotypic analysis of plants
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:

1. Master aspectic technique and tissue culture;
2. Use the Agrobacterium system to introduce a foreign gene, the β -glucuronidase gene (gusA) of E. coli, into the cells of tobacco;
   
3. Take advantage of the totipotency of plant cells and use these transformed cells to regenerate genetically engineered plants;
   
4. Confirm that the regenerated plants are transformed by
   
   1. measuring the activity of the introduced enzyme, β–glucuronidase (GUS) spectrophotometrically and histochemically;
   2. 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: Assaying the Transgenic Plants: DNA extraction, PCR, GUS by histochemistry
Lab 9: RE digestion, Agarose gel electrophoresis of the PCR product
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