Team:ZJUT-China/Design2018
Team:ZJUT-China
Light Control System Improvement Plan
1.Introduction of primary light control system
The primary light control system is shown in figure 1. Under dark conditions, the phosphate group is transferred from Yf1 protein to FixJ protein, the phosphorylated FixJ protein activates the PfixK2 promoter and then activates the downstream gene expression. When induced by light, the phosphorylation of Fixj protein will be blocked, and the expression of genes regulated by PfixK2 promoters will be inhibited.
2.Improvement plan of light control system
After being cultured at 37℃ for 24 to 48 hours in darkness, the expression of eGFP protein is not very high, showing our light control system lacks efficiency. So we wanted to improve the light control system and had a discussion with the team members. We came up with the following two plans: 1. PJ23100 promoter was replaced by PT7 promoter. 2. Replace the original E.coli DH5-αhost with another host bacteria.
2.1.Replacement of a strong promoter
We planned to replace the PJ23100 promoter with the PT7 promoter to reconstruct the light control system (Fig. 2), and determine the relationship between the culture time and the expression of eGFP protein.
2.2.Replacement of the host bacteria
he host of the primary light control system was E.coli DH5α. We suspected that the low expression of eGFP protein might be related to the host bacteria. Therefore, we planned to introduce the primary light control system plasmids that we've built into other host bacteria. (e.g. BL21, WT, MG1655, etc.), and select the best host(Fig. 3).
Sequential cutting
The goal of our project is to eliminate the resistance genes carried on the plasmids of the bacteria themselves. The method of elimination is to cleave the target resistance gene by light-controlled expression of the Cas9 protein through the guidance of sgRNA. So we designed the following gene circuits:
Our goal is to eliminate the resistance genes carried on the plasmids used in laboratories and fermentation plants, but it is not realistic to build our system on this plasmid because our system is huge. So we will need a second plasmid as a vector for the light-controlled cleavage system. In this case we need to remove the different resistance genes on the two plasmids, and their occurrence is in a certain order. To this end, we have improved the original design:
SgRNA1 will guide Cas9 to cleave the resistance gene on Plasmid 1, and sgRNA 2 guide Cas9 to cleave the resistance gene on Plasmid 2. When the resistance gene on Plasmid 1 is not cleaved, the plasmid is intact, and the normally expressed Repressor will repress the expression of sgRNA2; when the resistance gene on Plasmid 1 is cleaved, Repressor cannot be expressed normally, resulting in the expression of sgRNA. This led to the Cas9 cutting the resistance gene on Plasmid 2. Our design allows the two plasmids to be cleaved with a certain order to ensure self-destruction after elimination of the target gene.