BISC 219/2009:Creating a Transgenic Organism: Difference between revisions

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! 1
! 1
| 9/2 - 9/8 || set-up of plant transformation
| 9/8 - 9/14 || set-up of plant transformation
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! Independently
! Independently
|9/5 - 9/13
|9/11 - 9/17
|transfer of explants to selective medium; induce shoots
|transfer of explants to selective medium; induce shoots
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!5
!5
| 9/30 - 10/6
| 10/6 - 10/9
|transfer shoots to root inducing medium
|transfer shoots to root inducing medium
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|-
!7
!7
|10/15 - 10/21
|10/26 - 10/30
|transfer plantlets to soil
|transfer plantlets to soil
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!8
|11/9 - 11/13
|single leaf-disc PCR
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!9
!9
|11/12 - 11/18
|11/16 - 11/20
|GUS activity assays; set up PCR reactions
|GUS activity assays; run gel of PCR products
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!10
!10
|11/19 - 11/25
|11/30 - 12/4
|Gel electrophoresis of PCR product
|Data Analysis Workshop
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!11
!
|12/9
|12/11
|Plant Lab Report Due for all students
|Plant Lab Report Due for all students
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Revision as of 10:08, 24 June 2009

Wellesley College BISC 219 Genetics

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:

  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


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

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