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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, mutD[1]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 vector[2])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 Conception illustration of the working mechanism of mutD
Figure.2 rifampicin reversion mutants caused by mutD expression and the counts by agar plate

[1] Schaaper, R. M. Mechanisms of mutagenesis in the Escherichia-Coli mutator mutd5 - role of DNA mismatch repair. Proc. Natl. Acad. Sci. U. S. A. 85, 8126-8130,doi:10.1073/pnas.85.21.8126 (1988).

[2] Lou, C. B., Stanton, B., Chen, Y. J., Munsky, B. & Voigt, C. A. Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nature Biotechnology 30, 1137-+, doi:10.1038/nbt.2401 (2012).