Selection Of Mutants In Efficient Translocation Via Tat Translocation System In E.coli

The Tat translocation system plays important roles in Escherichia coli. The efficient translocation can be affected by multifold factors. In this research the method is afforded to separate the mutants of different efficient translocation that could be studied further more. The results revealed that the efficient translocation of ColV reporter protein could be showed by the growth of strains in liquid medium and on solid plate.Comparison the growth of various strains cultivated in liquid medium with solid medium, the result displayed that both of methods could show the efficient translocation. In order to do easy, strains were inoculated in solid medium firstly, and then were cultured in the liquid medium.Comparison the wild-type strain carrying RR-ColV(twin-Arg translocation) with KK-ColV (twin-Lys translocation) of the efficient translocation for ColV, the result showed that the strain carrying plasmid of RR-ColV could be killed severely. So the KK signal peptide was chosen in the experiment.The plasmid containing the genes of the colicinV and the Tat signal peptide was transfered into MC4100A, one of wild type strain of Escherichia coli, directly by the low energy nitrogen ion implantation. The plasmid was just used as system of the translocation-suicide probe to select out the mutants of different efficient translocation via Tat system. Moreover, the analysis for the characteristic array of the mutant cells, the growth in the medium of M9, the susceptibility to SDS, morphology characteristic and some biochemical characteristic were adopted further to identify the mutants that the efficiency were different.Ten mutants having different efficient translocation for ColV were singled out by above methods. Then they were cultured in the medium of M9 and the medium added 2% SDS respectively. At last two mutants were confirmed that the efficient to exporting were different to MC4100A×KK-ColV.