Team:Lambert GA/Demonstrate

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Demonstrate

To demonstrate the feasibility of regulated chromoprotein color expression dependent on the LacI- IPTG system, we received guidance and the preliminary genetic parts from Monica McNerney, a member of Georgia Institute of Technology, Dr. Styczynski’s laboratory. The supplied genetic sequence: pLambdaR LacI R0011 RFP (inducible promoter +Lac repressor + promoter + fluorescent protein reporter), and subsequent inductions of E. coli cells, that contained said plasmids, with incremental concentrations of IPTG, would have been an optimal demonstration of our projected constructs. Unfortunately we were not able to successfully visualize results. Methodology commenced with the transformation of standardized DH10-Beta cells with the RFP construct onto kanamycin and carbenicillin plates (we had received 1ng/uL minipreps o in separate kan and carb resistant expression vectors). Once the transformants had successfully grown for twentyfour hours we then streaked to completion, obtained a single colony the following day, and began running trials testing the viability of IPTG to indirectly induce RFP expression. The antibiotic-LB stocks, containing IPTG dilutions, were made on hand, each time the experiment began, in 50 mL storage flasks in order to preserve the quality of IPTG outside its standard storage temperature. Dilutions consisted of 0M, 10uM, 100uM, 500 Um and 1mM concentrations of IPTG. The trials were examined in triplicate where each trial consisted of 5mL aliquots of each IPTG solution in individual culture tubes; a fourth trial, the control, lacked cells and was solely composed of the cumulative IPTG concentrations in antibiotic-LB. In order to regulate the amount of cells placed in each experimental tube, 70uL of dH2O were pipetted into a microcentrifuge tube, and a single colony was then transferred and briefly vortexed to evenly distribute the cells; 5uL of dH2O-cell solution was aliquoted into each experimental tube. Once all culture tubes were correctly filled with their respective content, they were placed in that shaking-incubator for twenty four hours, finally to be removed, centrifuged and analyzed.
The first experiment produced unfavorable results where, although the cells fluoresced the correct “red” color, there was not a distinctive gradient associated with the incremental IPTG levels. Along with defective data, there was obvious contamination where each experimental tube contained arbitrary accumulations of black “dots” among the pellet. The experiment was redone and we determined the DH10-Beta we were supplied with had been outdated and was the cause of contamination. Despite renewing our supplies, subsequent experiments continued to elude hypothesized results as the experimental trials revealed no fluorescence. At this juncture, we chose to utilize the ATUM Protein Paintbox vectors with the IPTG Inducible T5 Promoter with separate TinselPurple (TsPurple) and ScroogeOrange, and Virginia Violet chromoprotein reporter sequences. Consequently, we repeated the above methodology and obtained expected results without unusual anomalies or contamination.
As hypothesized, there was an observable increase in color expression for both TsPurple and Scrooge Orange constructs directly correlated with increasing IPTG levels. The successful experiment was repeated 2 times for validity. Finally, the visible expression was then quantified using Lambert iGEM’s Chrome-Q, and complimenting, cellular app that determines HSL values of specified pictures. These results confirming the feasibility of our project and engineered machinery for the purpose of aiding underfunded labs.