Difference between revisions of "Part:BBa K3127998"

Revision as of 13:18, 22 September 2021

This part contains YF1, FixJ, PhlF, EsaI, TraR and CcdB.

This part contains YF1, FixJ, PhlF, EsaI, TraR and CcdB.This part contains YF1, FixJ, PhlF, EsaI, TraR and CcdB, which can release 30C12HSL and create CcdA protein with the exposure of the blue light.

Sequence and Features

Characterization

Figure 1/2 shows the trends of each bacteria in the LCBC-system. We got the data through the enzyme-labelled instrument and analyzed it to get the figure. The y-axis is the number of cells, and the x-axis is fluorescence intensity. There are two kinds of E.coli. One kind of E.coli carried sfGFP on the genome. The other kind of E.coli carried RFP on the genome. Cells were individually cultured in the LB medium for 12 hours before common inoculated in 12-well plates in LB medium in the first 13 hours. Figure 1 shows the different fluorescent intensity of E.coli under the light of 640-650 nm wavelength for the first 13 hours and the light of 460-465 nm wavelength for the next 12 hours.Figure2 shows the different fluorescent intensity of E.coli under the light of 640-650 nm wavelength for the first 13 hours and the light of 530-535 nm wavelength for the next 12 hours.

Figure 6: We can see that after 12 hours of blue light exposure, the number of E. coli bacteria carrying RFP dropped by half.

Figure 7: After 12 hours of green light exposure, the number of E.coli carrying GFP decreased by half.

Figure 3/4 shows the change in the ratio of two bacteria in LCBC system. We used CLSM to observe and process data. The second row shows the ratio of the two bacteria which have been exposed to the light of 640-650 nm wavelength for 12 hours and then to the light of 460-465 nm wavelength for the same time. The third row shows the proportion of the two bacteria which have been under the light of 640-650 nm wavelength for 12 hours and the light of 530-535 nm wavelength for another 12 hours.

Contribution from iGEM21-FAFU-China

Due to the pandemic, we did not have enough time to characterize and optimize the components used. There is a widely used set of photocontrol elements in prokaryotes. We contacted 2019 SDU -- China and obtained the element: BBa_K3127998

When many teams build these components, they often fail to control expression well. On the other hand, through preliminary characterization, we found that the target gene still had a certain amount of expression under dark conditions. Therefore, we consulted relevant literature to find possible causes and corresponding solutions, hoping to provide help to future iGEM teams.

It is pointed out in the literature that the instability of this light-controlled system is most likely due to the high expression level of LacI in chassis organisms. Under this premise, even the use of repressors with higher intensity than cI cannot solve the problem of low GFP expression level in darkness.

By adding degradation labels to LacI, the half-life of LacI can be significantly reduced, thereby increasing GFP expression in the dark. However, this can also increase GFP expression under light. Therefore, it is necessary to introduce lacO sequence downstream of PFixK2 promoter to control cI expression. This design creates a positive feedback loop, which severely represses GFP expression under light conditions.

PBBA uses IPTG-induced promoter, and GFP expression levels are basically the same under light and dark conditions.

Reference

[1] Lalwani MA, Ip SS, Carrasco-López C, Day C, Zhao EM, Kawabe H, Avalos JL. Optogenetic control of the lac operon for bacterial chemical and protein production. Nat Chem Biol. 2021 Jan;17(1):71-79. doi: 10.1038/s41589-020-0639-1. Epub 2020 Sep 7. PMID: 32895498.