Many challenges impeding genetically engineered bacteria from benefiting us can be attributed to the growth rate (e.g. infections in microbial therapies) and can be solved if we can better control over it. However, previous methods for growth rate control has many disadvantages: limited application scenarios, cell function disorders, etc. Here, we developed a novel system for precise growth rate control, by using dCas9 to target the DNA replication origin. Such system is highly tunable with multiple inputs, large dynamic range and non-detectable leakage. It functions in a gentle and reversible way without harming cell activities. Furthermore, we explored the potential of replication control in synthetic biology, including control of plasmid copy number and gene expression variation. Finally, we tried to design a safe therapeutic E. coli with high targeting specificity and controllable treatment intensity, promising to reduce the infection risk, which shows the broad application prospects of our system.
The function of our system is to slow down bacterial replication by combining dcas9 with oriC. On this basis, we also designed many different gene circuits to expand the function of our system.
We have carefully characterized the engineered bacteria we designed, found that they can work in the way we expected, and explored their potential functions.
We set up a series of models to quantitatively describe and analyze our experiment results. Our models can explain all kinds of phenomena observed in our experiment. They can explain the working principle of our system well.