Team:US AFRL CarrollHS/Design
Design
Our project uses a two-part circuit to create a simple and cost-effective test strip for determining whether hexavalent chromium is present in water. This test strip would act similar to a litmus test in its appearance and the response it gives. If the test strip is placed in water contaminated with chromium, the strip will change color to inform the user.
Concept design of working test strip
Our team was able to find a past iGEM team, iGEM Bielefeld 2015, who had worked with chromium detection. Their concept was to take a repressor, ChrB, which will bind to a corresponding promoter, ChrP, and inhibit the production of a reporter, in their case GFPa1 was used. However, in the presence of hexavalent chromium, the ChrB repressor will fall off the ChrP promoter and allow the reporter to be produced and thus giving a signal that the chromium is present.
The first step was to create the two-part circuit similar to that of iGEM Bielefeld from 2015. The first piece of the circuit is expressing the repressor, ChrB. We cibstitutively expressed ChrB from a T7 promoter in a pET11a backbone. ChrB expression could easily be tested by running a protein gel and determining whether the protein was being produced by the cell. The next piece of the circuit contains the promoter, ChrP, and the reporter, GFPa1. This also could be easily tested using the fluorescence from the GFPa1 as it will always be produced without the presence of the repressor.
However, after talking with a coatings specialist at Wright-Patterson Air Force Base, we were informed that this type of test could idealy be implemented as a quick home test. Therefore, an easier way of visualizing the results would likely be needed. In order to do this, bacterial flavin containing monooxygenase (bFMO) was used as the reporter as it produces an indigo color which can easily be seen without any aid of a machine. This achieves both goals of creating an easy to use test and one that is cost effective as no extra equipment is needed to read the results.
Repressor gene inhibiting production of bFMO
Chromium is present, repressor falls off and the reporter is produced
Later, we found out that chromium is toxic to cells. If the cells with our circuit were introduced to chromium, no response would be found as all the cells would die off. This caused our team to re-design our system of expression in a cell-free system. The cell-free system would not be affected by the toxicity of chromium as it is not alive. This would also reduce concern with using live bacteria in a home test.
Since the use of bFMO and cell free systems were new to the team and the lab, two positive controls were created; one containing GFPa1 and the other bFMO, each constitutively expressed from a T7 promoter. These were used in determining whether we could successfully express the reporters in a cell-free system. In addition to testing out cell free system in liquid, we also explored putting our cell free system on paper as well as lyophilizing and rehydrating the cell free systems. These platforms will make our system more fieldable.