Improve

BBa_K2789001 over BBa_K608102

This year we have improved the part BBa_K608102 designed by the 11-Freiburg iGEM team. This is a light control component Ccas. We want to use this component, but there are illegal EcoRI and SpeI sites in the sequence that do not comply with the RFC10 standard. Since this component needs to be used with another part Ccar, there will be problems during the assembly process. This team also encountered this problem, and this part has not been characterized.

In order to solve this problem, we re-synthesized the entire sequence (including Ccas and Ccar), optimized the codons, eliminating all the restriction sites that do not meet the RFC10 standard, and assembled our improved pathway BBB_K2789001 Subsequently, we used the promoter PcpcG+GFP, which is regulated by our part to characterize it.

Finally, we proved that Ccas can work in this pathway—it can be activated by green light, thus phosphorylate the Ccar and then turn on the downstream expression of the PcpcG promoter. This process can be ceased by red light as stated in the literature.

BBa_K608102（2262bp）—（codon optimized to avoid the restriction site that involved in RFC10 standard）—BBa_K2789009(2262bp) —（add Ccar to make it a complete functional unit）—BBa_K2789001(3416bp,submitted)

Characterization:

Experiments:

1. Transform the plasmids we have constructed into E.coli BL21 and select the single colony to cultivate in 10ml LB medium overnight.
2. Transfer 1ml medium after cultivating overnight into the 9ml sterilization LB medium
3. Cultivate the medium for 2 hours in 37℃
4. Add the IPTG to activate the gene expression
5. Get samples of medium after cultivating in 0h, 2.5h and 5h . Totally 3 samples each plasmid. Measure the fluorescence intensity of these samples ( activated with 480 nm light and measure in 530nm light)
6. After induced for 5 hours, cultivating the transformed E.coli under green light, red light and darkness for 2 hours.
7. Get samples after activated by different lights and then transform the green light into red light, vice versa , but bacteria in darkness keep dim, 8. Get samples after transforming the light for 1 hour.

Results:

We transformed the improved part BBa_K2789001 and use PcpcG promoter + GFP, to test the whole light control system, you can see from the picture that after expressing our part for 5h, it can express GFP after exposed to green light. And this process can be ceased after exposed to red light.