Vibrio natriegens can propagate in 10mins and has been applied in metabolic engineering, protein production as well as other fields. Until now, no efficient strategy for gene regulation on V. natriegens has been developed.
In our project, V. natriegens "Marionette", we develop a new strategy for strict and subtle gene regulation aiming at promoting the use of V. natriegens in synthetic biology. By introducing artificial promoters, insulators, UTRs, controllable ORI, and Marionette collection, we build a golden-gate-based part collection, which enables strict and tunable gene expression in V. natriegens. Furthermore, we engineered miniature CasΦ(751aa) and AsCas12f1(422aa) taking high sensitivity of V. natriegens to burden caused by protein expression, to achieve efficient CRISPRa and CRISPRi of multiple genes. We also explore the possibility to build incoherent feed-forward loops (iFFLs) by Cas protein.