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	9/2 - 9/8	set-up of plant transformation
Independently	9/5 - 9/13	transfer of explants to selective medium; induce shoots
5	9/30 - 10/6	transfer shoots to root inducing medium
7	10/15 - 10/21	transfer plantlets to soil
9	11/12 - 11/18	GUS activity assays; set up PCR reactions
10	11/19 - 11/25	Gel electrophoresis of PCR product
11	12/9	Plant Lab Report Due for all students

Experimental Objectives:

In this semester long experiment you will:

1. use the Agrobacterium system to introduce a foreign gene, the β -glucuronidase gene (gusA) of E. coli, into the cells of tobacco
   
2. take advantage of the totipotency of plant cells and use these transformed cells to regenerate genetically engineered plants
   
3. confirm that the regenerated plants are transformed by
   
   1. measuring the activity of the introduced enzyme, β–glucuronidase (GUS) spectrophotometrically and histochemically
   2. PCR to test directly for the presence of the introduced genes and
      
4. 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