Team:Nanjing High School/Contribution
Contribution
Contribution
During our project, we have developed an existing part (BBa_K1065303). In former experiments, the part consists of coding sequence YF1, response regulator FixJ, and inducible promoter controlled by pFixK2. It is an intermediate part to build a blue-light-sensitive circuit.
(Fig.1 The sequence of the existing Blue Sensor.)
Former teams use FixK2 promoter (BBa_K592006) to make YF1 be able to regulate FixJ, the blue light sensing protein.
(Fig.2 The sequence of FixK2 promoter which is used by the existing Blue Sensor.)
In our experiments, we changed this promoter with J23100 promoter (BBa_J23100)and T7 
terminator. This reconstruction of Blue Sensor makes it to function after being induced, which enables us to record time with other genes when there is blue light.
(Fig.3 The sequence of our developed Blue Sensor.)
The developed Blue Sensor enables later iGEM teams to use with a broader application. For J23100 promoter is a constitutive promoter, it can be used to tune the expression level. Moreover, restriction sites on the Blue Sensor make it easier for later teams to modify and develop the plasmid related to their own projects.
Validated contribution
During our project, we have designed a new part to record the time E.coli is exposed to blue light. Cas1-Cas2 is a special protein that can catch fragments. In our experiment, we transferred the plasmid with this protein to the micro-organism so that when the micro-organism is placed under blue light, the promoter will function and the protein will then begin to catch fragments. After a certain amount of time, gel analysis we did to examine the result showed that samples under blue light had a shorter strip, which meant Cas1-Cas2 effectively caught and stored the fragments.
(Fig.1 The function of Cas1-Cas2 catching system without and with blue light.)
In this light-sensitive experiment, the composition of FixK2-Cas3, T7-Cas1-Cas2, and J23100-BlueSensor is shaken at 37 ℃ for 12 hours. We induced the bacteria liquid with L-Arabinose and IPTG. After well mixing the bacteria liquid, we burnt the liquid at 95 ℃. Then we operated PCR to examine whether Cas1-Cas2 caught the fragments by analyzing whether the strip is longer than the one without blue light.
(Fig.2 The gel analysis of whether Cas1-Cas2 protein expresses after induced by L-Arabinose.)
There are many ways in which later iGEM teams can use this part. For example, in an immunity system, Cas1 and Cas2 will form a stable complex, and when a foreign DNA fragment invades the cell, Cas1-Cas2 will catch the fragment, integrate it into the CRISPR array, and form a memory, which indicates that if a team is going to introduce a immunity system against some certain diseases, Cas1-Cas2 can help a lot. Moreover, since it can catch fragments under several circumstances, a team which wants a protein to limit the expression of certain genes can use Cas1-Cas2. In conclusion, Cas1-Cas2 is of great help not only to our team, but also to later iGEM teams.