Part:BBa_K3127998
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 3869
Illegal NheI site found at 3892 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 4808
Illegal XhoI site found at 3712 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 587
Illegal NgoMIV site found at 659
Illegal NgoMIV site found at 749
Illegal NgoMIV site found at 767
Illegal NgoMIV site found at 1259
Illegal NgoMIV site found at 1552
Illegal NgoMIV site found at 1646
Illegal AgeI site found at 301
Illegal AgeI site found at 1427
Illegal AgeI site found at 3655 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1316
Illegal BsaI site found at 5068
Illegal BsaI.rc site found at 200
Illegal SapI.rc site found at 3127
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 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.
OptoLAC1, OptoLAC2 and OptoLAC3 were fused with different degradation tags after LacI, and we could obviously observe the difference in the expression level of GFP in light and darkness, and at the same time meet the target of high expression of target gene in darkness, and almost no expression of target gene in light.
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.
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