Isolation Mutants Of Tat From Escherichia Coli By The Low Energy Nitrogen Ion Beam Implantation

In this thesis, the technique of the low energy nitrogen ion beam implantation was used to mutate the wild type strain of Escherichia coli.in order to get mutants whose Tat protein pathway had been blocked. Seven transformants were get after transformed p8760 into strain MC4100A implanted by nitrogen ion beam. They were different from strain MC4100A. For example, transformants, cell array in line, while MC4100A, cell is single. The former grow weakly on 2% SDS plate and can,t grow in M9 medium anaerobicly. So some important characters of these seven strains were same with tat mutant, such as B1LKOA. But they also have different properties with B1LKOA. These mutants grow slowly at 37℃ while they can form colony of diameter about 0.5mm at 4 ℃ after three days. These results demonstrated that the site of mutation may relate with function of Tat pathway. In order to test whether the mutation is same with tatA,tatB,tatC and tatE or not, several strains were eliminated plasmid of p8760 and were transformed into plasmid of p8737. We observed that their changed characters can,t recover to the wild type,s. This result confirm that mutation was occur independly in tat gene. A gene library of the genomic DNA of MC4100A was tried to reconstruct for the sake of finding the site of mutation. This genomic DNA was purified and then broken randomly into fragments that are the correct size for cloning into the chosen vector of pBR322. Now we are choosing the proper enzyme to broke the genomic DNA. This research confirms that the ion implantation can induces gene transform into cells and causes mutagenesis. Meanwhile, we isolated a mutant that was tat-like strain, but it was different with the Tat pathway we have known. So we can certain there is still have other component participate in the Tat transport system. Surely our work established the base of perfecting it,s constitutes and creating model mechanism of Tat transport system.