molecular analysis of transgenic biofuel plantsmolecular analysis of transgenic biofuel plants...
TRANSCRIPT
![Page 1: Molecular Analysis of Transgenic Biofuel PlantsMolecular Analysis of Transgenic Biofuel Plants NSF-REU Program Summer 2011 Joe Meisenbach. Molecular Techniques Learned • Reviving](https://reader033.vdocuments.net/reader033/viewer/2022053020/5f2b21b516db6e49ee499285/html5/thumbnails/1.jpg)
Molecular Analysis of Transgenic Biofuel PlantsNSF-REU Program
Summer 2011Joe Meisenbach
![Page 2: Molecular Analysis of Transgenic Biofuel PlantsMolecular Analysis of Transgenic Biofuel Plants NSF-REU Program Summer 2011 Joe Meisenbach. Molecular Techniques Learned • Reviving](https://reader033.vdocuments.net/reader033/viewer/2022053020/5f2b21b516db6e49ee499285/html5/thumbnails/2.jpg)
Molecular Techniques Learned
• Reviving Bacterial Cell Cultures from Glycerol Stocks
• Setting Up Overnight Cultures for Agrobacterium transformations
• Plasmid DNA Extraction• Transformation of E. coli
/Agrobacterium via Electroporation
• Preparation of Glycerol Stocks• Plant DNA Extractions
![Page 3: Molecular Analysis of Transgenic Biofuel PlantsMolecular Analysis of Transgenic Biofuel Plants NSF-REU Program Summer 2011 Joe Meisenbach. Molecular Techniques Learned • Reviving](https://reader033.vdocuments.net/reader033/viewer/2022053020/5f2b21b516db6e49ee499285/html5/thumbnails/3.jpg)
Molecular Analyses of Putative Transgenic Materials
![Page 4: Molecular Analysis of Transgenic Biofuel PlantsMolecular Analysis of Transgenic Biofuel Plants NSF-REU Program Summer 2011 Joe Meisenbach. Molecular Techniques Learned • Reviving](https://reader033.vdocuments.net/reader033/viewer/2022053020/5f2b21b516db6e49ee499285/html5/thumbnails/4.jpg)
Constituents of a PCR Reaction Plant Genomic DNA dNTPs
(deoxynucleotide triphosphates) Primers
(Forward and Reverse) Taq Polymerase MgCl2
Buffer Nuclease Free Water
Basic PCR Diagram
![Page 5: Molecular Analysis of Transgenic Biofuel PlantsMolecular Analysis of Transgenic Biofuel Plants NSF-REU Program Summer 2011 Joe Meisenbach. Molecular Techniques Learned • Reviving](https://reader033.vdocuments.net/reader033/viewer/2022053020/5f2b21b516db6e49ee499285/html5/thumbnails/5.jpg)
Polymerase Chain Reaction
• In PCR, a three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules.
• The reaction mixture is heated to denature (separate) the DNA strands.
• The mixture is cooled to allow annealing (hydrogen bonding) of short, single-stranded DNA primers complementary to sequences on opposite sides at each end of the target sequence.
• A heat-stable DNA polymerase extends the primers in the 5′3′ direction.
![Page 6: Molecular Analysis of Transgenic Biofuel PlantsMolecular Analysis of Transgenic Biofuel Plants NSF-REU Program Summer 2011 Joe Meisenbach. Molecular Techniques Learned • Reviving](https://reader033.vdocuments.net/reader033/viewer/2022053020/5f2b21b516db6e49ee499285/html5/thumbnails/6.jpg)
Standardization of Annealing Temperatures for PCR
• Objective: Standardization of Annealing Temperatures for various primers that we intend to use for PCR reactions
• Genes Standardized:• Beta-glucuronidase (GUS)• Green Fluorescent Protein (GFP)• Cold Binding Factor 3 (CBF3)• H10N7
• S-Adenosyl methionine Decarboxylase (y.SAMdc )• To standardize the Annealing Temperature for
the PCR, the same PCR was run under different temperatures.
![Page 7: Molecular Analysis of Transgenic Biofuel PlantsMolecular Analysis of Transgenic Biofuel Plants NSF-REU Program Summer 2011 Joe Meisenbach. Molecular Techniques Learned • Reviving](https://reader033.vdocuments.net/reader033/viewer/2022053020/5f2b21b516db6e49ee499285/html5/thumbnails/7.jpg)
Results; Standardization of Annealing Temperatures for PCR
Lane
1
Lane
2
Lane
3
Lane
4
Lane
5
Lane
6
Lane
7
Lane
8
Gene: Beta-glucuronidase (GUS) Lane Number 1 2 3 4
Temperature 49.7 ºC 50.3 ºC 51.9 ºC 54.3 ºC
Intensity (I-%) 81.78 77.47 79.2 80.72
Lane Number 5 6 7 8
Temperature 57.1 ºC 60.3 ºC 63.6ºC 1 kb K Ladder
Intensity (I-%) 79.51 74.52 71.5 -
Optimal Annealing Temp: 49.7 ºC
![Page 8: Molecular Analysis of Transgenic Biofuel PlantsMolecular Analysis of Transgenic Biofuel Plants NSF-REU Program Summer 2011 Joe Meisenbach. Molecular Techniques Learned • Reviving](https://reader033.vdocuments.net/reader033/viewer/2022053020/5f2b21b516db6e49ee499285/html5/thumbnails/8.jpg)
Results; Standardization of Annealing Temperatures for PCR (cont.)
Gene: H10N7 Lane
1
Lane
2
Lane
3
Lane
4
Lane
5
Lane
6
Lane
7
Lane
8
Lane Number 1 2 3 4
Temperature 49.7 ºC 50.3 ºC 51.9 ºC 54.3 ºC
Intensity (I-%) - - - -
Lane Number 5 6 7 8
Temperature 57.1 ºC 60.3 ºC 63.6ºC 1 kb Ladder
Intensity (I-%) 35.71 83.38 59.87 -
Optimal Annealing Temp: 60.3ºC
![Page 9: Molecular Analysis of Transgenic Biofuel PlantsMolecular Analysis of Transgenic Biofuel Plants NSF-REU Program Summer 2011 Joe Meisenbach. Molecular Techniques Learned • Reviving](https://reader033.vdocuments.net/reader033/viewer/2022053020/5f2b21b516db6e49ee499285/html5/thumbnails/9.jpg)
Results; Standardization of Annealing Temperatures for PCR (cont.)
Gene Optimal Annealing Temperature (ºC)
GUS 50
H10N7 60
CBF3 50
eGFP 52
y. SAMdc 51
![Page 10: Molecular Analysis of Transgenic Biofuel PlantsMolecular Analysis of Transgenic Biofuel Plants NSF-REU Program Summer 2011 Joe Meisenbach. Molecular Techniques Learned • Reviving](https://reader033.vdocuments.net/reader033/viewer/2022053020/5f2b21b516db6e49ee499285/html5/thumbnails/10.jpg)
Standardization of DNA Quantity to be Utilized for PCR
Concentration of DNA: 1µg/µL
Lane
1
Lane
2
Lane
3
Lane
4
Lane
5
Lane
6
Lane
7
Lane
8
Gene: Enhanced Green Fluorescent Protein (eGFP)
Concentrations: 1µg/µL, 500, 50 and 5 ng/µL
Concentration with greatest yield: 1µg/µL
![Page 11: Molecular Analysis of Transgenic Biofuel PlantsMolecular Analysis of Transgenic Biofuel Plants NSF-REU Program Summer 2011 Joe Meisenbach. Molecular Techniques Learned • Reviving](https://reader033.vdocuments.net/reader033/viewer/2022053020/5f2b21b516db6e49ee499285/html5/thumbnails/11.jpg)
PCR Results of Putative Transgenic Plants
Gene: Beta-glucuronidase (GUS) Plant: Jatropha La
ne 1
Lane
2
Lane
3
Lane
4
Lane
5
Lane
6
Lane
7
Lane
8
Lane # 1 2 3 4
Description -ve Control
+ve Control
Jatropha Plant 1
Jatropha Plant 2
DNA Intensity
0 54.56 0 69.7
Lane # 5 6 7 8
Description Jatropha Plant 3
Jatropha Plant 4
Jatropha Plant 5
1 Kb Ladder
DNA Intensity
0 0 72.55 -
![Page 12: Molecular Analysis of Transgenic Biofuel PlantsMolecular Analysis of Transgenic Biofuel Plants NSF-REU Program Summer 2011 Joe Meisenbach. Molecular Techniques Learned • Reviving](https://reader033.vdocuments.net/reader033/viewer/2022053020/5f2b21b516db6e49ee499285/html5/thumbnails/12.jpg)
PCR Results of Putative Transgenic PlantsGene: S-Adenosyl methionine Decarboxylase (SAMdc)Plant: Tomato
Lane
1
Lane
2
Lane
3
Lane
4
Lane
5
Lane
6
Lane
7
Lane
8
Lane # 1 2 3 4
Description -ve Control
+ve Control
Tomato Plant 1
Tomato Plant 2
DNA Intensity
0 + 0 +
Lane # 5 6 7 8
Description Tomato Plant 3
Tomato Plant 4
Tomato Plant 5
1 Kb Ladder
DNA Intensity
0 0 + -
Positive Control
Tomato Sample 1
Tomato Sample 5
![Page 13: Molecular Analysis of Transgenic Biofuel PlantsMolecular Analysis of Transgenic Biofuel Plants NSF-REU Program Summer 2011 Joe Meisenbach. Molecular Techniques Learned • Reviving](https://reader033.vdocuments.net/reader033/viewer/2022053020/5f2b21b516db6e49ee499285/html5/thumbnails/13.jpg)
My NSF-REU Summary• Reviving bacterial cultures
– Streaking, Setting up overnight cultures for plant transformations• Electropoartion of E.coli / Agrobacterium with new plasmids• Preparation of Glycerol stocks• DNA Extractions
– Plasmid; Total plant genomic DNA• PCR
– Gradient PCR, Regular PCR to standardize the DNA quantity to be used in a PCR reaction
• Analyses of putative transgenic plants– Standard PCR– Gel Electrophoresis
![Page 14: Molecular Analysis of Transgenic Biofuel PlantsMolecular Analysis of Transgenic Biofuel Plants NSF-REU Program Summer 2011 Joe Meisenbach. Molecular Techniques Learned • Reviving](https://reader033.vdocuments.net/reader033/viewer/2022053020/5f2b21b516db6e49ee499285/html5/thumbnails/14.jpg)
What I have Learned during this Internship
• Preparation of Glycerol Stocks
• Labeling Tubes, Recordkeeping and Inventory
• Setting Up Overnight Cultures and maintenance of vectors for the entire laboratory
• PCR
![Page 15: Molecular Analysis of Transgenic Biofuel PlantsMolecular Analysis of Transgenic Biofuel Plants NSF-REU Program Summer 2011 Joe Meisenbach. Molecular Techniques Learned • Reviving](https://reader033.vdocuments.net/reader033/viewer/2022053020/5f2b21b516db6e49ee499285/html5/thumbnails/15.jpg)
Acknowledgements• National Science Foundation for the fellowship and
research opportunity.• Penn State Harrisburg for facilitating this through the
brand new Biotechnology Laboratory.• Dr. Shobha Potlakayala • Dr. Sairam Rudrabhatla • Matt Reitzel: student mentor• Nasie Constantino and Krysta Haggins• Swati Patel and Aneel Maini for assisting me with all
my work • Alison Shuler and Julie Dauber: program coordination