Team:BGIC-Union/Improve
Improvement
Previously, Peking University 2015 created a PC report system by connecting a split luciferase with a dCas9 protein.{N-luc-dCas9(BBa_K1689010) and C-luc-dCas9(BBa_K1689009) }
Figure 1. Schematic of the paired dCas9 (PC) Reporter system of Peking University. A complex is formed by dCas9, split luciferase(Nluc or Cluc) and sgRNA. In the presence of target DNA, each of the complexes will bind to the specific sites indicated by sgRNA. When they approach to each other, the split luciferase will reassemble and generate bioluminescence signal.
This year, we improve this PC report system by substituting the split luciferase with split T7 RNA polymerase.{N-t7-dCas9(BBa_K2371000) and C-t7-dCas9(BBa_K2371001)} Our design has two major advantages over Peking University’s project. First, we made various types of output possible. Because the reassembled protein is an RNA polymerase, it can convert ctDNA into different types of signals, including GFP, RFP, luciferase or LacZ. Besides, with the presence of Target sequence, reunion T7 polymerase can transcribe DNA into mRNA which will be further translated into protein consistently. Therefore, the signal can be magnified over time even if the initial ctDNA concentration is undesirable.
Figure 2. The schematic illustration of paired dCas9 system(2). With the presence of target DNA, each complex will bind with pre-designed sgRNA-binding site on the sequence. When the split T7 polymerase approach each other close enough, they will become active and start transcription by adding report gene with T7 promotor in cell free system. After transcription, the mRNA will be translated into report protein like GFP and RFP which generate signal output.
Also we create two report gene pT7-eforRed(BBa_K2371011) and pT7-amilGFP(BBa_K2371012) to produce output in our PC report system. We add a T7 promotor and a RBS in front of the coding sequence of report protein in order to improve it to be utilized in our paired dCas9 T7 report system in cell free system.
Figure 3. The plasmid of pT7-eforRED. pT7-eforRED is composed of a T7 promotor, an RBS and the coding sequence of RFP. This plasmid is used to act as report gene in our report system.
Figure 4. The plasmid of pT7-amilGFP. pt7-amilGFP is composed of a T7 promotor, an RBS and the coding sequence of GFP. This plasmid is used to act as report gene in our report system.
Reference
1.Yihao,Z. et al.,2017.Paired Design of dCas9 as a Systematic Platform for the Detection of Featured Nucleic Acid Sequences in Pathogenic Strains. ACS Synth. Biol., 2017, 6 (2), pp 211–216. 2.Tiyun,H. et al.,2016.Engineered photoactivatable genetic switches based on the bacterium phage T7 RNA polymerase.ACS Synthetic Biology,http://pubs.acs.org on October 31, 2016.
