Molecular Analysis of Transgenic Biofuel Plants. NSF-REU Program Summer 2011 Joe Meisenbach
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1 Molecular Analysis of Transgenic Biofuel Plants NSFREU Program Summer 211 Joe Meisenbach
2 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
3 Molecular Analyses of Putative Transgenic Materials
4 Basic PCR Diagram Constituents of a PCR Reaction Plant Genomic DNA dntps (deoxynucleotide triphosphates) Primers (Forward and Reverse) Taq Polymerase MgCl2 Buffer Nuclease Free Water
5 Polymerase Chain Reaction In PCR, a threestep 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, singlestranded DNA primers complementary to sequences on opposite sides at each end of the target sequence. A heatstable DNA polymerase extends the primers in the 5 3 direction.
6 Standardization of Annealing Temperatures for PCR Objective: Standardization of Annealing Temperatures for various primers that we intend to use for PCR reactions Genes Standardized: Betaglucuronidase (GUS) Green Fluorescent Protein (GFP) Cold Binding Factor 3 (CBF3) H1N7 SAdenosyl methionine Decarboxylase (y.samdc ) To standardize the Annealing Temperature for the PCR, the same PCR was run under different temperatures.
7 Lane Number Temperature 49.7 ºC 5.3 ºC 51.9 ºC 54.3 ºC Intensity (I%) Lane Number Temperature 57.1 ºC 6.3 ºC 63.6ºC 1 kb K Ladder Intensity (I%) Optimal Annealing Temp: 49.7 ºC Lane 8 Lane 7 Lane 6 Lane 5 Lane 4 Lane 3 Lane 2 Gene: Betaglucuronidase (GUS) Lane 1 Results; Standardization of Annealing Temperatures for PCR
8 Lane Number Temperature 49.7 ºC 5.3 ºC 51.9 ºC 54.3 ºC Intensity (I%) Lane Number Temperature 57.1 ºC 6.3 ºC 63.6ºC 1 kb Ladder Intensity (I%) Optimal Annealing Temp: 6.3ºC Lane 8 Lane 7 Lane 6 Lane 5 Lane 4 Lane 3 Gene: H1N7 Lane 2 Lane 1 Results; Standardization of Annealing Temperatures for PCR (cont.)
9 Results; Standardization of Annealing Temperatures for PCR (cont.) Gene Optimal Annealing Temperature (ºC) GUS 5 H1N7 6 CBF3 5 egfp 52 y. SAMdc 51
10 Lane 8 Lane 7 Lane 6 Lane 5 Lane 4 Lane 3 Lane 2 Gene: Enhanced Green Fluorescent Protein (egfp) Lane 1 Standardization of DNA Quantity to be Utilized for PCR Concentrations: 1µg/µL, 5, 5 and 5 ng/µl Concentration with greatest yield: 1µg/µL Concentration of DNA: 1µg/µL
11 Lane # Description ve Control +ve Control Jatropha Plant 1 Jatropha Plant 2 DNA Intensity Lane # Description Jatropha Plant 3 Jatropha Plant 4 Jatropha Plant 5 1 Kb Ladder DNA Intensity Lane 8 Lane 7 Lane 6 Lane 5 Lane 4 Lane 3 Lane 2 Gene: Betaglucuronidase (GUS) Plant: Jatropha Lane 1 PCR Results of Putative Transgenic Plants
12 Lane # Description ve Control +ve Control Tomato Plant 1 Tomato Plant 2 DNA Intensity + + Lane # Description Tomato Plant 3 Tomato Plant 4 Tomato Plant 5 1 Kb Ladder DNA Intensity + Tomato Sample 5 Tomato Sample 1 Positive Control Lane 8 Lane 7 Lane 6 Lane 5 Lane 4 Lane 3 Lane 2 Gene: SAdenosyl methionine Decarboxylase (SAMdc) Plant: Tomato Lane 1 PCR Results of Putative Transgenic Plants
13 My NSFREU 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
14 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
15 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
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