BISC 219/2009:Creating a Transgenic Organism: Difference between revisions
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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. | 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'''== | |||
{| border="1" | |||
|+ | |||
! Lab # !! Dates !! Activity | |||
|- | |||
! 1 | |||
| 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 | |||
|9/11 - 9/17 | |||
|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]] | |||
|- | |||
!4 | |||
| 9/29 - 10/5 | |||
|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 | |||
|10/26 - 10/30 | |||
|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 | |||
|11/11 - 11/17 | |||
|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 | |||
|11/18 - 11/24 | |||
|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 | |||
|11/30 - 12/4 | |||
|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> | ||
#Master aspectic technique and tissue culture; | |||
#Use the Agrobacterium system to introduce a foreign gene, the β -glucuronidase gene (gusA) of E. coli, into the cells of tobacco;<br> | |||
#Take advantage of the totipotency of plant cells and use these transformed cells to regenerate genetically engineered plants;<br> | |||
#Confirm that the regenerated plants are transformed by<br> | |||
##measuring the activity of the introduced enzyme, β–glucuronidase (GUS) spectrophotometrically and histochemically; | |||
##using PCR to test directly for the presence of the introduced genes. | |||
{| border="1" | |||
|+ | |||
! 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 | |||
|} | |||
[[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
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 |
9 | 11/11 - 11/17 | Restriction enzyme digest of PCR reactions Agarose gel electrophoresis of digested PCR |
10 | 11/18 - 11/24 | Spectrophotometric GUS enzyme activity assay Leaf Extract Preparation Spectrophotometric Assay for GUS activity |
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:
- Master aspectic technique and tissue culture;
- Use the Agrobacterium system to introduce a foreign gene, the β -glucuronidase gene (gusA) of E. coli, into the cells of tobacco;
- Take advantage of the totipotency of plant cells and use these transformed cells to regenerate genetically engineered plants;
- Confirm that the regenerated plants are transformed by
- measuring the activity of the introduced enzyme, β–glucuronidase (GUS) spectrophotometrically and histochemically;
- 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