Contribution to part: BBa_K1231000

Pasr promoter is a pH-sensitive promoter that can trigger transcription under an acidic environment, and it was the key factor that regulated our proton pump’s expression level. This promoter was proposed by Suziedeliene et al. in 1999 and first used for iGEM by iGEM13_Northwestern. According to the results that appear on the iGEM, most of the team monitors their promoter function by incubating the E. coli cells with Pasr promoter that is linked to a GFP protein in different pH values of M9 medium. However, GFP intensitive was sensitive to pH value. According to G. Doherty et al. research result in 2010, GFP is more sensitive to pH change than mCherry, which increases its intensity from pH4.5 to 7.5 under the same condition. This could be because of a huge error in recording the transcription level of the promoter’s function. As a result, our team this year chose to use the mCherry fluorescent signal, instead of GFP, to become our protein expression label.

Fig. 1 indicated the result of our experiment this year. In this experiment, we used the initial average fluorescence signal of each sample to calculate the fold changes. According to Fig. 2a, the Pasr- promoter functioned remarkably at pH 4.5 and pH 5 M9 medium. The signal increased significantly after 2 hours and the signal was about three times higher contrasted with the other three pH values (at 5 hours). The fluorescence signal at pH5.5, pH6, and pH7 remained unchanged. All this data gave a complete pH-sensitive image for Pasr promoter.

The inward proton pump: Xenorhodopsin:

This year, we have introduced a new part into the iGEMThe light-driven inward proton pump XeR (BBa_K4357000) is derived from xenorhodopsin in Nanosalina spp. It is a seven-transmembrane alpha-helix protein that functions with its cofactor called retinal(chromophore). When there is a light source applied to the protein, the conformational change of the retinal will induce inward proton pumping of bacteria and generate an electrochemical proton gradient across the membrane. In most of the previous studies, scholars are researching action potentials that react on neural cells; this kind of mechanism only occurs within a milli-seconds. In our studies, instead of doing short term light induce experiment, we perform a long term protein functional assay for the XeR protein, which will become some useful data for the iGEMers after years

Reference

1. Doherty, G.P., Bailey, K., & Lewis, P.J. (2010). Stage-specific fluorescence intensity of GFP and mCherry during sporulation In Bacillus Subtilis. BMC Research Notes, 3, 303 - 303.

2. Shevchenko, V., Mager, T., Kovalev, K., Polovinkin, V., Alekseev, A., Juettner, J., Chizhov, I., Bamann, C., Vavourakis, C., Ghai, R., Gushchin, I., Borshchevskiy, V., Rogachev, A., Melnikov, I., Popov, A., Balandin, T., Rodriguez-Valera, F., Manstein, D. J., Bueldt, G., Bamberg, E., … Gordeliy, V. (2017). Inward H⁺ pump xenorhodopsin: Mechanism and alternative optogenetic approach. Science advances, 3(9), e1603187. https://doi.org/10.1126/sciadv.1603187

3. Shevchenko, V., Mager, T., Kovalev, K., Polovinkin, V., Alekseev, A., Juettner, J., Chizhov, I., Bamann, C., Vavourakis, C., Ghai, R., Gushchin, I., Borshchevskiy, V., Rogachev, A., Melnikov, I., Popov, A., Balandin, T., Rodriguez-Valera, F., Manstein, D. J., Bueldt, G., Bamberg, E., … Gordeliy, V. (2017). Inward H⁺ pump xenorhodopsin: Mechanism and alternative optogenetic approach. Science advances, 3(9), e1603187. https://doi.org/10.1126/sciadv.1603187