Synthetic biology is based on standard parts, and characterizing specific parts can save future users time studying each necessary component when building a system. Inter-laboratory work can help characterize parts more comprehensively using different materials and methods. This year, we measured the creation of:
We measured the following components we created:
1.Protein rational design：BBa_K5319667
2.Sensitivity analysis of salicylic acid-induced expression：
Psal BBa_K5319686
nahR-Q168R BBa_K5319685
3. Sensitivity analysis of C4-HSL-induced expression：
PRhlR BBa_J61051
RhlR BBa_C0171
After constructing the vioE two-site mutation library, we hoped to specifically characterize the enzyme activity of different mutants vioE. In order to simplify the experimental process, our scheme finally selected to directly detect the pigmentation production ability of E.coli introduced with different mutants vioE to represent the enzyme activity after one iteration.
The results showed that the activity of different mutants was different from that of wild type vioE.
After combining the enzyme activity prediction model of the software, we focused on repeating some mutants that fell in the hot spots of enzyme activity, and obtained and verified mutants that increased their activity by up to 30%.
To ensure that the salicylic acid and C4-HSL based orthogonal quorum sensing system we designed is sufficient to regulate the pigment production for cloth printing and dyeing requirements, We use sfGFP as a reporter gene to simulate the salicylic acid and C4-HSL induced expression, and the sensitivity was analyzed.
We construct the promoters corresponding to RhlR and nahR in front of sfGFP, which are induced by C4-HSL and salicylic acid respectively. Meanwhile, we construct a plasmid containing corresponding receptors. We co-transfer the two plasmids into K12 for sensitivity analysis.
We use fluorescent microplates to detect fluorescence intensity at different signaling molecule concentrations. The results show that the fluorescence intensity increase with the concentration of salicylic acid and C4-HSL in a certain range. The measured data can be used to guide us to regulate the induction time at the production end to achieve different color printing patterns.
(1)VioE mutants of different mutants have uneven catalytic capacity. By combining the software model and wet experimental data, we obtained vioE mutants with strong enzyme activity.
(2)the fluorescence intensity increase with the concentration of salicylic acid and C4-HSL in a certain range, and the response of salicylic acid induced expression is more sensitive. However, when the concentration of salicylic acid is above 2.6e-4M, the fluorescence intensity decrease. It may be due to the inhibition of E. coli growth caused by high concentration of salicylic acid.
