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 FAFU-China 2021
Overview
There is a set of light control components widely used in prokaryotes（ YF1, FixJ, PhlF）. We got in touch with 2019 SDU-China and got the component: BBa_K3127998 (https://parts.igem.org/Part:BBa_K3127998).By referring to relevant literature, we found that a not gate (LacI and LacO) can be added on the basis of BBa_K3127998 (YF1, FixJ, PhlF,GFP) to extend its application. Green fluorescent protein can be expressed under dark conditions, but not under light conditions. The extended function of BBa_K3127998 (YF1, FixJ, PhlF,GFP) meets the requirements of our project this year, and we hope it can help iGEM teams in the future.
(Note: BBa_K3127998 (YF1, FixJ, PhlF,GFP) is different from the repressor used in the references, but its function and principle are basically the same. CI is used instead of PhlF for convenience of description.)
Characterization
We transformed the plasmid carrying BBa_K3127998 (YF1, FixJ, PhlF,GFP) into E. coli DH5α, and then selected the correct transformants in the chloramphenicol resistance plate. Pick a single colony and inoculate it into a resistant medium for overnight culture. Pick a small amount of bacterial liquid with an inoculation needle and streak it on a resistant plate, and culture for 2 days under light (natural light) and dark conditions.
Using a fluorescent stereo microscope to observe a single colony, the results showed that the colony cultured under dark conditions had weak fluorescence, and the colony cultivated under light conditions had strong fluorescence, which was consistent with the characterization results of 2019 SDU-China. At the same time, we also proved that white light can be used instead of blue light to induce BBa_K3127998 (YF1, FixJ, PhlF,GFP).
Analyze
Adding non gates (LacI and LacO) directly to BBa_K3127998 (YF1, FixJ, PhlF,GFP) may make the optical control system unstable. To solve this problem, we looked at the relevant literature.
The reason is most likely that the expression of LacI in chassis organisms is too high. In this case, the use of repressors with higher intensity than cI cannot solve the problem of low GFP expression in the dark.
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.
We believe that the above optimization can significantly improve the stability and efficiency of the optical control system.
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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