Team:Lambert GA/Demonstrate
Demonstrate
To demonstrate the feasibility of 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: p-lambda-r LacI R0011 RFP, transformed into E. coli cells, and then induced with incremental concentrations of IPTG, would have been an optimal demonstration of our projected constructs. Unfortunately, we were not able to successfully visualize results. Therefore, 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 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 potency of IPTG when indirectly inducing RFP expression. The antibiotic-LB stocks, containing cumulative IPTG concentrations, were made on-site (each time the experiment began) in 50 mL disposable centrifuge tubes in order to preserve the quality of IPTG outside its standard storage temperature. Concentrations of IPTG consisted of 0M, 10uM, 100uM, 500 uM and 1mM where the solvent was antibiotic-LB. The trials were examined in triplicate where each trial consisted of 5mL aliquots of each IPTG solution in individual culture tubes (five tubes/ trial); a fourth, control trial 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, 95uL of diH2O were pipetted into a microcentrifuge tube, and a single colony was then transferred and briefly vortexed (one or two "touches") to evenly distribute the cells; 5uL of diH2O-cell solution was aliquoted into each experimental tube. The 95uL of diH2O measurement accounted for the 5uL placed into each experimental tube, as well as any unintentional error when inoculating liquid cultures. Once all culture tubes were correctly filled with their respective content, they were placed in the shaking-incubator for twenty four hours, then centrifuged and analyzed.
![](https://static.igem.org/mediawiki/2017/e/e7/T--Lambert_GA--ExpressionLabSetup.jpeg")
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 incoherent data, there was obvious contamination where each experimental tube contained arbitrary accumulations of black “dots” in 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, continued experimentation revealed no fluorescence and, therefore, eluded hypothesized results. At this juncture, we chose to purchase and utilize the ATUM Protein Paintbox expression vectors with the IPTG Inducible T5 Promoter with separate TinselPurple (TsPurple),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 all three chromoprotein constructs directly correlated with increasing IPTG levels. The successful experiment was repeated three times for validity. Finally, the visible expression was then quantified using the Lambert iGEM’s Chrome-Q and complimenting, mobile app that determines HSV values of specified pictures. These results confirmed the feasibility of our project and engineered machinery for the primary target of aiding underfunded labs in synthetic biology research.
