Team:Tsinghua-E/Part1

Part 1: THU-E Mutation Part

A plasmid used for the construction of high-diversity library in vivo ingenome level. In this vector, highly error-prone dnaQ mutant, mutD1 was cloned downstream of araBAD promoter to control the mutation rate of the target genome by the concentration of araBAD promoter’s inducer, L-arabinose, in a strict manner.E. Coli JM109 carrying different vectors of pBAD_B0030-mutD-sfGFP, pBAD_B0032-mutD-sfGFP and pBAD_SDA_RBS-mutD-sfGFP(this RBS sequence was derived from the RBS sequence upstream of sfGFP in original AraC_pBAD_CI_OR222-sfGFP vector2)were constructed. By detecting the induced fluorescence intensity, we found that pBAD_B0030-mutD-sfGFP, andpBAD_SDA_RBS-mutD- sfGFPhave relatively higher mutD expression. The increaseof mutation rate induced by our mutation part was measured by quantifying the reversion of rifampinresistance caused by mutation in genome.pBAD_SDA_RBS-mutD- sfGFPcould increase the genome mutation rate up to 10 times compared with negative control with 1g/L induction concentration of L-arabinose.

Figure.1 plasmid map for THU-E mutation part

Figure.2 rifampicin reversion mutants caused by mutD expression and the counts by agar plate

Figure.3 Conception illustration of the working mechanism of mutD