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Step 1: Enzyme digest to remove stop codon from original plasmid Step 2: Mutagenesis PCR to remove stop codon Step 3: Mutagenesis PCR to remove 1071 G.

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Presentation on theme: "Step 1: Enzyme digest to remove stop codon from original plasmid Step 2: Mutagenesis PCR to remove stop codon Step 3: Mutagenesis PCR to remove 1071 G."— Presentation transcript:

1 Step 1: Enzyme digest to remove stop codon from original plasmid Step 2: Mutagenesis PCR to remove stop codon Step 3: Mutagenesis PCR to remove 1071 G mutation CONSTRUCTION OF THE FUSION PROTEIN Run gel to verify DNA presence and band size Starting Point: Original pcDNA3.1 V5-His plasmid Final Product: pcDNA3.1 human GPR120 V5/HIS 6 fusion protein Amp (R) ApaI(323) Neo(R) 6609 bp ApaI(1142) V5 epitope 6xHis Steps 1-4 The specific objective of this research was accomplished through the steps below. HEK1ug3ug5ug 3 rd Western Blot- HEK V5/His Actin Blot- To confirm protein concentration HE K 1ug3u g 5u g 1st Western Blot- HEK V5/His HEK 3ug 5ug HEK3ug5ug 2 nd Western Blot- HEK Strip The newly created fusion protein for the V5/HIS 6 was verified using 1µg, 3µg, and 5µg of the fusion protein DNA concentrations. The 5µg concentration DNA looks less than the 3µg DNA concentration, while it should appear thicker so an Actin Blot was performed. The findings were supported with the actin blot. The blot showed much less protein loaded into the gel due to the smaller actin band in the 5µg. When stripped and B- actin stained, band length in unknown concentration was the same as 3µg. Correct Orientation Reverse Orientation An enzyme digest was performed to remove the stop codon using HindIII and Not1 The stop codon was not successfully removed, so an alternative method was performed, site-directed mutagenesis PCR (see Step 2). Step 4: Validation using Western and Actin Blots Star t Sto p TAA Start AUG Stop TAA V5/His 6 Sto p Start Stop V5/His 6 Sto p Actual Result Expected Result: G The site directed mutagenesis PCR was performed to remove the stop codon. The stop codon was removed, however there was a mutation in the sequence at 1071G. A specific primer was used to perform another site- directed mutagenesis PCR to remove the mutation. Successfully removed the 1071 G mutation There was no stop codon before the V5/HIS 6 tag on the tail end of the sequence. The 1071G was removed from the sequence correctly with a specially designed primer. The stop codon came after the V5/HIS 6 tag. Star t AUG Stop TAA V5/His 6 Sto p Primer G Actual Result: Expected Result: Start Stop V5/His 6 Stop Codon Removed INTRODUCTION Overall Purpose: To develop a novel mechanism to stimulate GLP-1 and downstream insulin secretion. Immediate Purpose: To assess the potency and efficacies of various free fatty acids (FFA) in stimulating GLP-1 secretion through GPR120. Rationale: Diabetes is a growing epidemic in the United States, as well as a global health threat. Diabetes is a disease in which the body does not produce enough insulin properly. Background Information: Glycolipid peptide or GLP-1 is a potent incretin hormone. This hormone enhances the glucose-dependent secretion of insulin in beta cells (Hirasawa, 2005). Free fatty acids provide energy and also serve as signaling molecules in the secretion of incretin peptides. GPR120 is produced in the intestines and functions as a receptor for long chain free fatty acids. The diagram above demonstrates how GPR120 works in the human body through the following four steps: Step 1- Intake of food Step 2- The free fatty acid, FFA, is used for the GPR120 to move into the intestines Step 3- The increased amount of free fatty acids allows the insulin secreting peptide, GLP1, to become regulated Step 4- Insulin secretion increases and becomes regulated Specific Research Objective: To find the novel mechanism to stimulate GLP-1 and downstream insulin secretion by utilizing molecular biological techniques to engineer a V5/HIS 6 epitope onto the tail end of the cloned human GPR120 receptor CONCLUSIONS An enzyme digest was performed to remove the stop codon in the Human GPR120 pcDNA 3.1. The stop codon was not removed, so a site directed mutagenesis PCR was removed to attempt to remove the stop codon. The stop codon was removed; however, a mutation at 1071G was evident in the sequence. To remove the 1071G mutation, a primer was designed to remove this using site- directed mutagenesis PCR. The mutation was removed with the stop codon coming after the V5/HIS 6. A western blot was performed to verify the protein concentration in the fusion protein. The stop codon was successfully removed yielding a fused cDNA. The expression of the fused cDNA translates to fused protein. The western blot showed much less protein loaded into the gel due to the smaller actin band in the 5µg. This was validated by the actin blot, which confirmed the protein concentration in all wells. Accomplished the specific research objective by successfully constructing a fusion protein that may increase GLP-1 secretion in the body. SIGNIFICANCE Although further research is required the fusion protein constructed in this study may increase GLP-1 secretion in the body. This could eliminate diabetic dependence on artificial insulin by : Balancing levels of glucagon Regulate insulin homeostasis Regulate food intake and satiety Results could be use to develop novel anti-diabetic agents which target GPCRs Could also treat other disorders such as obesity FUTURE RESEARCH The Next Step: Current experimentation in the laboratory includes transfecting the fusion protein constructed in this study into human embryonic kidney (HEK) cells. This cell system will be used to assess free fatty acid binding through radioligand binding assays, G protein signaling by assessing activation of G proteins, non-G proteins will be done by assessing free fatty acid mediated phosphorylation of ERK. The above process will illustrate whether or not GLP-1 secretion is mediated by the free fatty acids. Future experimentation will include in vivo testing using rats or mice and seeing response by the mouse to GLP-1. CONCLUSIONS SIGNIFICANCE FUTURE RESEARCH CONSTRUCTION OF THE FUSION PROTEIN INTRODUCTION

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