Design Our whole plan is to control the cell chromosome replication dynamically (Figure 1). After investigation, we decided to combine the CRISPR and optogenetic tools to construct our system. Figure 1. The magic idea of our project. Like mentioned in Description, DnaA protein is essential for cell cycle control$^{[1]}$, so we decide to interrupt its binding to the corresponding DNA using CRISPR/dCas9. Under the guidance of gRNA, dCas9 will bind to where DnaA should bind, blocking further reactions of forming replisome$^{[2]}$. Without a replisome, the chromosome can’t replicate, stucking in phase B. Compared with engineering DnaA protein, this method is easier and more robust, and readily integrated into other prokaryotes. Optogenetic tools are used to regulate the function of dCas9 due to its specificity in time and space and the easiness of its manipulation, and meanwhile it is also considered to be a proper connection between an organism and a computer. So we decided to use light as a medium to control the whole system. To accomplish such a system, we designed two approaches; one is based on the regulation of transcription level, and the other is based on protein interaction. On the transcription level, we used the CcaS-CcaR system, which is developed from cyanobacteria and is well used in synthetic biology$^{[3]}$. The CcaS-CcaR system is a two-component system. Under the green light, CcaS protein will be phosphorylated and the CcaR protein accept this phosphate and dimerize into a transcriptor inducing the transcription of gRNA, leading to the inhibition of replication. In the red light, the gRNA will stop transcription and degrade in a short time, and the inhibition will decrease in a short time, freeing cells from blocking (Figure 2). Corresponding results are HERE. Figure 2. The CcaS-CcaR system is developed from Synechocystis PCC 6803 and engineered into E. coli. It is a two component system (TCS) in which CcaS can sense green light and autophosphorylate as a membrane-binding protein and CcaR can be phophorylated by CcaS-P and dimerize into a transcription factor.
The protein level of light-controlled dCas9 system is based on the split protein and light induced dimerization (LID) protein. By infusing split dCas9 and LID protein together, dCas9 can be controlled by light (Figure 3). The pMag and nMag developed from fungal is chosen to induce the complement of dCas9$^{[4]}$. They are engineered VVD proteins using FAD as its light sensing molecule. Under the irritation of blue light, the conformation change of FAD influences the structure of pMag and nMag, exposing its dimerization domain. We choose this pair of proteins due to their low molecular weights and tunable dynamics (Figure 3). Figure 3. The working mechanism of dCas9-fused pMag and nMag under blue light. These two approaches both can satisfy our need in a certain way, but the former one is simpler and better coordinated with the metabolic state of the chassis, and the latter one is more difficult to fulfill. We tried two approaches at the same time. For more information please view EXPERIMENTS.
References 1. Mott,M.L. and Berger,J.M. (2007) DNA replication initiation: mechanisms and regulation in bacteria. Nat. Rev. Microbiol., 5,343–354. 2. Wiktor, J., Lesterlin, C., Sherratt, D. J., & Dekker, C. (2016). CRISPR-mediated control of the bacterial initiation of replication. Nucleic Acids Res, 44(8), 3801-3810. 3. Fernandez-Rodriguez, J., Moser, F., Song, M., & Voigt, C. A. (2017). Engineering RGB color vision into Escherichia coli. Nature Chemical Biology, 13(7), 706-708. 4. Kawano, F., Suzuki, H., Furuya, A., & Sato, M. (2015). Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins. Nat Commun, 6, 6256.