The Construction And Study Of A New Mobile Recombineering System Of PYM-Red

Recombineering is a new developed genetic engineering technique in recent years. Compared to traditional genetic engineering technology, recombineering requires no restriction endonucleases and ligase. With linear DNA fragment amplified by PCR as targeting molecular, it can precisely modify the bacterial chromosome or BAC/PAC vectors which carried large molecular genome DNA sequence. The novel technology provides an exciting method for gene functional researching, bacterial genetics and vaccine researching.A long fragment from exo to cI857 of phage X was inserted into the low-copy pACYC184 plasmid by Gap-repair - a method of in-vivo cloning - and constructed a new high-efficient recombination plasmid named pYM-Red. In this system, Red recombination enzyme of λphage is restrictedly regulated just as DY330 strain. Furthermore, the plasmid can be transferred to different host and be widely applied. The recombination efficiency of pYM-Red system is 5 -fold and 6-fold higher than pBR322-Red and pKD46 plasmid respectively.Three bacterial mutation strains with deletion of part sequence of Pl operator of DY330 were constructed using the method of ssDNA recombination to study the correlation between the regulation and function of Red recombination enzyme. Compared to the wild-type DY330, the recombination efficiency of the three mutations was decreased. In order to increase the recombination efficiency, a new recombination plasmid named pYM-RedT was constructed by knocking the transcription terminator TL3 into the downstream of the Red gene of pYM-Red plasmid. Results showed that the recombination efficiency of pYM-RedT increased about 2 folds than that of pYM-Red system.The pGEX4Tl-Exo and pET28a-Exo prokaryocyte-expressing plasmids were constructed to study the effect of the Red recombination enzyme production had on...