Molecular Analysis of Transgenic Biofuel PlantsNSF-REU Program

Summer 2011Joe Meisenbach

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

Molecular Analyses of Putative Transgenic Materials

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

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.

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.

Results; Standardization of Annealing Temperatures for PCR

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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

Results; Standardization of Annealing Temperatures for PCR (cont.)

Gene: H10N7 Lane

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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

Results; Standardization of Annealing Temperatures for PCR (cont.)

Gene Optimal Annealing Temperature (ºC)

GUS 50

H10N7 60

CBF3 50

eGFP 52

y. SAMdc 51

Standardization of DNA Quantity to be Utilized for PCR

Concentration of DNA: 1µg/µL

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Gene: Enhanced Green Fluorescent Protein (eGFP)

Concentrations: 1µg/µL, 500, 50 and 5 ng/µL

Concentration with greatest yield: 1µg/µL

PCR Results of Putative Transgenic Plants

Gene: Beta-glucuronidase (GUS) Plant: Jatropha La

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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 -

PCR Results of Putative Transgenic PlantsGene: S-Adenosyl methionine Decarboxylase (SAMdc)Plant: Tomato

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Lane # 1 2 3 4

Description -ve Control

+ve Control

Tomato Plant 1

Tomato Plant 2

DNA Intensity

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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

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

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

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