Team:SCU-China/Engineering

SCU-China

1. We achieved successful dCasphi-based CRISPRa in Vibrio.natriegens

We firslt apply protein structure analysis to find the best activation domain for CRISPRa, the results showed that the α domain of RNAp from Vibrio natriegens could accomplish the best gene activation compared with others.

So we firstly fusion V.nαdomain in the N domain of dCasΦ，but it impeded the gene expression while classic AD SoxS showed a successful activation. We assumed that this may due to the fusion manner which was then proved by both docking analysis and practical function tests that Cas-linker-AD is better than our first design, AD-linker-Cas.

Moreover, by comparing the modeling results from the I-TASSER server, we selected a relatively more appropriate linker from the six linkers collected.

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Fig 1. Integration of the protein structure model and our design.

2. We decrease we leakiness of LacIAM-Ptac in Vibrio.natriegens by a RNA regulator

We observer that the leakiness of LacIAM-Ptac in Vibrio.natriegens is extremely strong and nearly performs as a constitutive promoter.

So we applied a RNA regulator to arrest the translation of mSarlet-I so as to decrease the leakiness in the translation level.

3. Our strain produced violacein and naringenin successfully

Team:SCU-China/Engineering

Engineering success

CRISPRa, Improvement of LacIAM in V.n and metabolic engineering