Difference between revisions of "Team:HZAU-China/Design"

Design Our whole plan is to control the cell replication dynamically. After research, we decided to combine the CRISPR and optogenetic to construct our system. Like mentioned in description, DnaA protein is essential for cell cycle control, so we decide to interrupt its binding to corresponding DNA using CRISPR/dCas9. Under the guidance of gRNA dCas9 will bind to where DnaA should bind blocking further reaction to form replisome. Without replisome, the chromosome can’t replicate block it at phase B. Compared with engineering DnaA protein, this method is more easy and robust, and can be readily integrated into other prokaryotes. Optogenetic is used to regulate the function of dCas9 due to its specificity in time and space and its easy manipulation, and it is also considered to be a proper connection between organism and computer. So we decided to use light to control the whole system. To achieve this system, we also designed two approach, one is based on the reaction of transcription level, and the other is based on the protein interaction. On the transcription level, we used CcaS-CcaR system, which is developed from cyanobacteria and is well used in synthetic biology. The CcaS-CcaR system is a two-component system. Under the green light, the phosphorylation of CcaS protein will happen and the CcaR protein will accept this phosphate and dimerize into a transcriptor inducing the transcription of gRNA, in which 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 cell from blocking. Corresponding results please see HERE. The protein level of light controlled dCas9 system is based on the split protein and light induced dimerization protein. By infusing split dCas9 and LID protein together can make dCas9 under the control of light( fig). The pMag and nMag developed from fungal is chosen to induce the complement of dCas9. They are engineered VVD protein 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 revealing its dimerization domain. We choose this pair of protein due to its low molecular weight and tunable dynamics. For split dCas9, we not only tried the traditional way to split, but also tried a split method based on its structure and function(fig. ) These two approaches both can satisfy our need in a certain way, but the former one is simpler but more related to metabolic state of chassis, and the latter one is more difficult to fulfill. So we tried two approaches at the same time. For more information please view EXPERIMENT.