BASIL2022GV3R8E: Difference between revisions
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Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization. | Once we felt confident enough to finalize our substrate hypothesis, we began testing in vitro. Beginning with bacterial protein overexpression and affinity chromatography, we were able to purify our POI and begin testing with real substrates. Below are the results of our Uncoupled Kinase Assay, reported in terms of specific activity (mg/mL). Our results from this assay further supports our idea of protein 3r8e assisting in the phosphorylation of glucose. A total of five hexose substrates were tested in vitro, detailed in the table below. Based on these results, we were able to strengthen our initial hypothesis and continue characterization. | ||
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For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein. | For further validation, we conducted an SDS analysis and provided below is the gel image. Indicated by the black box is our POI, around 34 kDa. Results were not as clear as anticipated, and in future studies, we would need to utilize different chromatography methods to yield higher quality protein concentrations and conduct a pre and post induction to visualize the purity of our protein. | ||
