BISC 219/2009:Creating a Transgenic Organism

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Wellesley College BISC 219 Genetics

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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
Set-up of the tranformation
Independently 9/11 - 9/17 transfer of explants to selective medium; induce shoots
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
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
Leaf Extract Preparation

Spectrophotometric Assay for GUS activity
Calculations

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: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