Team:Lambert GA/Demonstrate



Demonstrate


To demonstrate the feasibility of chromoprotein color expression, dependent on the LacI-IPTG system, Lambert iGEM 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: pλR LacI R0011 RFP, transformed into E. coli cells, and then induced with incremental concentrations of IPTG, would have been an optimal demonstration of the projected constructs. Unfortunately, results could not be successfully visualized. Therefore, methodology commenced with the transformation of standardized DH10-Beta cells with the RFP construct onto kanamycin and carbenicillin plates (received 1ng/uL minipreps in separate kan and carb resistant expression vectors). Once the transformants had successfully grown for 24 hours they were streaked to completion, a single colony was obtained the following day, and trials were run 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 (15 tubes x 5uL/tube), as well as for any unintentional error when inoculating liquid cultures (extra 20uL of diH2O-cell solution present in microcentrifuge tube). 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.



Proof of Concept Expression Lab Setup

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 it was determined the supply of DH10-Beta cells had been outdated and was the cause of contamination. Despite renewing laboratory and biological materials, continued experimentation revealed no fluorescence and, therefore, eluded hypothesized results. At this juncture, it was resolved to purchase and utilize the ATUM Protein Paintbox with the IPTG Inducible T5 Promoter with separate TinselPurple (tsPurple), ScroogeOrange, and VirginiaViolet chromoprotein reporter sequences. Consequently, the above methodology was repeated and expected results were obtained without unusual anomalies or contamination.



Natalie Shih hard at work in the lab.

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 the project and engineered machinery for the primary target of aiding underfunded labs in synthetic biology research.


ATUM Protein Paintbox Expression Results

Chrome-Q System Protocol

1. Make 5mL liquid cultures in triplicate of cumulative concentrations of IPTG in LB (0mM, 10uM, 100uM, 500uM, 1mM)
2. Make one trial (5 tubes) of control group containing 5mL of LB
3. Fill microcentrifuge with [(5 x #of experimental tubes) + 20] uL of diH2O (extra 20uL of diH2O accounts for unintentional error when inoculating liquid cultures)
4. Inoculate the microcentrifuge with a visible amount of cells from cultured plate (cells contain IPTG inducible sequence with color-expressive reporter)
5. Briefly (one or two “touches”) vortex microcentrifuge to suspend and evenly distribute cells in diH2O
6. Micropipet 5uL of suspended diH2O-cell mixture into each experimental tube (this step ensures a regulated amount of cells/ experimental tube)
7. Place in incubator at 37C for 24 hours
8. After growth period, centrifuge at 2500 RCF for ten minutes to obtain a pellet
9. Micropipet 200 uL of pellet into a PCR tube and centrifuge for two minutes in an electrically-powered centrifuge or 3 minutes in the 3D-fuge
10. Vortex lightly (one or two “touches”) and Pipette up and down to resuspend cells.
11. Take 20uL from the center of the resuspended cells and transfer to a new PCR tube. (to continue with the 20uL, skip to the paragraph below; keep the remaining 180uL of cells for later directions).
12. Take 20uL from the center of the resuspend cells and transfer to a new PCR tube
13. Dilute 20uL of suspended cells from each tube in 80uL of H20.
14. Perform a serial dilution to get down to a .000X concentration. (There should be 4 dilutions).
15. Plate 20uL of cells from the final dilutions in a lawn in order to get individual countable colonies across the plate.
16. Grow cells in the incubator overnight.
17. Form a grid on the plate and use a random number generator to select a grid section.
18. Count the number of cells in the grid section.
19. Average the number of cells counted per microliter for each dilution and average the three. This will result in the average number of cells per microliter: the optical density.
20. Dividing the the HSL values by the optical density results in the quantification measurements.
21. (Proceeding with the remaining 180uL of cells) Centrifuge the remaining 180uL of cells and plate 160uL of the supernatant. Avoid the pellet.
22. Pipette up and down in the remaining supernatant to resuspend.
23. Take 17uL of the sample and plate in triplicates.
24. Place the plates in the incubator (37C) for 24 hours
We performed three serial dilutions of 100uL of TinselPurple. We then plated 20uL of this on a quadrant of a plate. We determined the concentration of the cells was too great, due to the formation of lawns as opposed to countable colonies, and that a fourth dilution at a .000X will generate the most desirable growth. We also determined that each dilution did need to be plated in a lawn format on individual plates.

















Set Up for Dilutions of Tinsel Purple Triplicates















Set Up for Dilutions of Tinsel Purple Triplicates

















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