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<br>[[BISC 219/2009: Mod 3 Experiment 1 Creating the transgenic plant | Set-up of the tranformation]]
|-
|-
! 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<br>[[BISC 219/2009: Mod 3 Experiment 1 Creating the transgenic plant Days 3-5 | Selecting for transformants]]
|-
|-
!5
!4
| 9/30 - 10/6
| 9/29 - 10/5
|transfer shoots to root inducing medium
|transfer shoots to root inducing medium<br> [[BISC 219/2009: Mod 3 Experiment 1 Creating the transgenic plant Week 4-5 | Transfer to rooting medium]]<br>
|-
|-
!7
!7
|10/15 - 10/21
|10/26 - 10/30
|transfer plantlets to soil
|transfer plantlets to soil<br> [[BISC 219/2009: Mod 3 Experiment 1 Creating the transgenic plant Week 8| Tranfering the Plantlets to soil]]<br>
|-
!8
|11/4 - 11/10
|single leaf-disc PCR<br>
<font color=green>[[BISC 219/2009: Mod 3 Structural Evidence for Transgenic Plants| Protocols for Structural Evidence for Transgenic Plants]]</font color=green><br>
Histochemical GUS enzyme activity assay<br><font color=green>[[BISC 219/2009: Mod 3 GUS Activity Assay by Histochemistry| Protocols for GUS Activity Assay by Histochemistry]]</font color=green><br>
|-
|-
!9
!9
|11/12 - 11/18
|11/11 - 11/17
|GUS activity assays; set up PCR reactions
|Restriction enzyme digest of PCR reactions<br>
Agarose gel electrophoresis of digested PCR<br><font color=green>[[BISC 219/2009: Mod 3 Experiment 3 Agarose gel electrophoresis of PCR| Protocols for RE digestion, Agarose gel electrophoresis of the PCR product]]</font color=green><br>
|-
|-
!10
!10
|11/19 - 11/25
|11/18 - 11/24
|Gel electrophoresis of PCR product
|Spectrophotometric GUS enzyme activity assay<br><font color=green>[[BISC 219/2009: Mod 3 Leaf Extract Preparation| Leaf Extract Preparation]] <br>
[[BISC 219/2009: Mod 3 Spectrophotometric Assay for GUS activity| Spectrophotometric Assay for GUS activity]] <br>
[[BISC 219/2009: Mod 3 Calculations| Calculations]]<br></font color=green><br>
Phenotypic analysis of plants
|-
|-
!11
!11
|12/9
|11/30 - 12/4
|Plant Lab Report Due for all students
|Data Analysis Workshop<br><font color=green>[[BISC 219/2009: Mod 3 Analyzing the data on the putative transgenic and control plants| Instructions for Analyzing the data on the putative transgenic and control plants]]</font color=green>
|-
! Dec 11
|12/11
|Plant Genetic Engineering Paper Due<br>for all students
|-
|}
|}


=='''Experimental Objectives:'''==
=='''Experimental Objectives:'''==


'''In this semester long experiment you will:'''  <br>       
'''In this semester long experiment you will:'''  <br>       
#use the Agrobacterium system to introduce a foreign gene, the β -glucuronidase gene (gusA) of E. coli, into the cells of tobacco<br>
#Master aspectic technique and tissue culture;
#take advantage of the totipotency of plant cells and use these transformed cells to regenerate genetically engineered plants<br>
#Use the Agrobacterium system to introduce a foreign gene, the β -glucuronidase gene (gusA) of E. coli, into the cells of tobacco;<br>
#confirm that the regenerated plants are transformed by<br>
#Take advantage of the totipotency of plant cells and use these transformed cells to regenerate genetically engineered plants;<br>
##measuring the activity of the introduced enzyme, β–glucuronidase (GUS) spectrophotometrically and histochemically
#Confirm that the regenerated plants are transformed by<br>
##PCR to test directly for the presence of the introduced genes and  <br>
##measuring the activity of the introduced enzyme, β–glucuronidase (GUS) spectrophotometrically and histochemically;
#become adept at sterile technique.  
##using PCR to test directly for the presence of the introduced genes.  




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|-
|-
| Effect of random gene insertion in a large genome
| Effect of random gene insertion in a large genome
| Bacterial pathogen:  ''Agrobacterium tumefaciens'' || Aseptic tissue culture
| Vector bacterium:  ''Agrobacterium tumefaciens'' || Aseptic tissue culture
|-
|-
| Plant totipotency
| Plant totipotency
|Tobacco plant: ''Nicotiana tobacum''
| Transgenic recipient: Tobacco plant- ''Nicotiana tobacum''
|DNA Extraction
|DNA Extraction
|-
|-
|Regulation of gene expression
|Regulation of gene expression
|gusA gene from ''E. coli''
|Transgene: gusA gene from ''Escherichia coli'' encoding beta-glucoronidase
|PCR
|PCR
|-
|-
|Genetic Engineering and creation of genetically modified organism
|Genetic Engineering and creation of genetically modified organism
|
| _
|Agarose gel electrophoresis
|Agarose gel electrophoresis
|-
|-
|Phenotypic Selection
|Phenotypic Selection
|
| _
|Transfection & selection by antibiotic resistance;
|Transfection & selection by antibiotic resistance;
Enzyme function assays: colorimetric & histochemical
Enzyme function assays: colorimetric & histochemical
|}
|}
[[BISC 219/2009: Mod 3 Background| Background]] <br>
[[BISC 219/2009: Mod 3 Experiment 1 Media Recipes | Transgenic Plants: Media Recipes]]<br>
[[BISC 219/2009: Mod 3 Experiment 1 Creating the transgenic plant | Lab 1: Creating the Transgenic Plant: Day 1]]<br>
[[BISC 219/2009: Mod 3 Experiment 1 Creating the transgenic plant Days 3-5 | Outside of Lab in the first week: Creating the Transgenic Plant: Days 3-5]]<br>
[[BISC 219/2009: Mod 3 Experiment 1 Creating the transgenic plant Week 4-5 | In Lab 4: Creating the Transgenic Plant: Week 4-5]]<br>
[[BISC 219/2009: Mod 3 Experiment 1 Creating the transgenic plant Week 8| In Lab 7-8: Creating the Transgenic Plant: Week 8]]<br>
[[BISC 219/2009: Mod 3 Experiment 2 Assaying the transgenic plants| Lab 8:GUS by histochemistry]]<br>
[[BISC 219/2009: Mod 3 Structural Evidence for Transgenic Plants| Lab 8:Structural Evidence for Transgenic Plants: DNA extraction, PCR]] <br>
[[BISC 219/2009: Mod 3 Experiment 3 Agarose gel electrophoresis of PCR| Lab 9: RE digestion, Agarose gel electrophoresis of the PCR product]]<br>
[[BISC 219/2009: Mod 3 Leaf Extract Preparation| Lab 10:Leaf Extract Preparation for GUS Spectrophotometric Assay]] <br>
[[BISC 219/2009: Mod 3 Spectrophotometric Assay for GUS activity| Lab 10:Spectrophotometric Assay for GUS activity]] <br>
[[BISC 219/2009: Mod 3 Calculations| Lab 10:Calculations]]<br>
[[BISC 219/2009: Mod 3 Analyzing the data on the putative transgenic and control plants| Lab 11: Analyzing the all the data on the putative transgenic and control plants]]<br>

Latest revision as of 05:01, 29 October 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
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

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