Team:FAFU-CHINA/Contribution
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 (http://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 meets the requirements of our project this year, and we hope it can help iGEM teams in the future.
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)[1].
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 thetarget
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.
Suggest
We have summarized the experience and lessons from the establishment of the team to the present, and hope to be helpful to the iGEM team in the future.
1. Experiment
①
The pre-design of the experiment is necessary. The team should hurry up to apply for the right to use the
laboratory and pre-purchase the required reagents in advance. Before entering the laboratory, all members of the
experimental team must be trained in safety and operation.
② Using blue light to induce microorganisms to express the target gene, it is best to cooperate with
electronic sensors to explore the induction conditions in advance. Too strong blue light will cause the death of
microorganisms, and too weak blue light has a poor induction effect.
2. Modeling
Our modeling work is divided into: mathematical modeling, protein modeling and simulation modeling of aroma diffusion. We believe that the core of modeling is to help the project solve some substantive problems. Modeling does not have to be bundled with experiments. The students in our modeling group also linked the model with our hardware.
3. Human practice
①
Human practical work is the best way to connect the project with reality. According to the results of human
practice, we will improve our projects step by step, so that the projects are truly beneficial and responsible
for the world.
② Before launching HP activities, be sure to study the local laws carefully and report to the relevant
departments in time. It is necessary to carry out HP activities correctly under the premise of legality and
ethics.
③ Because HP activities involve all aspects of communication, we encourage all groups of people to
assist the
HP group in carrying out human practice activities.
4. WiKi production
①
In the case of wiki code conflicts and unsuitability, you should promptly communicate with the wiki
producers of the previous participating teams to discuss solutions together.
② Locally produced webpages must be uploaded to the official website in time, and there will be
sufficient
time for debugging when errors occur.
③ We encourage all team members to assist web team members to upload pictures used in WiKi.
④ It is best to start writing WiKi one month in advance and update the content in WiKi in time. Don't
squeeze
your work into the last few weeks!
⑤ Igem wikis have a lot of default styles that need to be removed using CSS and JS to make it easier for member sites to write.
⑥There are too many pictures to upload, so the name of the picture should be standardized and easy to understand, and the address of the uploaded picture should be standardized and recorded, which will greatly facilitate the reference of pictures on the wiki page.
5. Team building
①
A truly united team is a team that coordinates, develops together and advances towards the same goal. Team
members must actively communicate with each other. In addition to regular group meetings, we also recommend that
future teams organize various forms of team building activities to enhance the relationship between members.
② The instructor is also a part of the team. We can seek advice and help from the teacher when
encountering
various problems, not just limited to the problems encountered in the laboratory.
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.
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