Cloning Of Pseudomonas Putida KT2440 And Corynebacterium Glutamicum ATCC 13032 Chromosome Gene By Recombinant Engineering

Recombineering (Red/ET, recombination mediated genetic engineering) a DNA cloning and modification technology based on the recombinase catalyzed homologous recombination, it is a highly efficient genetic engineering strategy. The recombinases used are mainly from the X phage origin redap genes or Rac prophage origin recET genes. By using recombineering, it is theoretically possible to knock-out and modify any chromosome DNA of bacteria or fungi, and recombineering avoids the gene cloning steps which are needed by other gene deletion means. The targeting DNA used in recombineering can be directly obtained from OE-PCR (Overlap extension PCR). Recombineering was firstly achieved in Escherichia coli, and since it has been extended to other bacteria and fungi. Pseudomonas putida KT2440 is an environmental organism that plays important roles in biodegradation and heterologous expression; Corynebacterium glutamicum ATCC 13032 is an industrial amino acid producing organism. With the completion of the genome sequencing of the two organisms, more and more study has been focused on the genes’ function. Gene deletion is an essential to study the function of gene and its encoded protein, and recombineering-mediated can be a highly efficient gene deletion strategy.This thesis introduces recombineering mediated gene deletion of P. putida KT2440 and C. glutamicum ATCC 13032. For P. putida KT2440, two-plasmid and one-plasmid markerless Cre/loxP gene deletion were realized in four gene deletion systems which we custructed. The method involved firstly preparation of the P. putida KT2440 Red-competent electrocompetent cells with the expression of recombinase, then electroporated into the OE-PCR generated linear targeting DNA. The transformation suspension was spread on LB agar plate containing kanamycin. In the two-plasmid gene deletion system, the Cre inducible-expression plasmid was transformed after validation of the genotype of the kanamycin resistant strain. The gene deletion efficiency can be up to 100%. In the one-plasmid system, The gene deletion efficiency of the first step only can be up to 1/5 to the two-plasmid gene deletion system, we speculate that may be associated with the high background levels of Cre. The kanamycin resistant strain was acetone-induced to eliminate the kanamycin resistance gene. Though the gene deletion efficiency of the one-plasmid system is less than that of two-plasmid system, former is more convenient and more suit to high-throughput manipulations.Redαβand recET recombineering system are constructed which contain recombinase gene and I-Scel mediated double stranded break repair mechanism was used for C. glutamicum ATCC 13032 gene deletion. The method involved the preparation of Red-competent cells and electroporation of OE-PCR generated linear targeting DNA. Under kanamycin selection, target gene deletion was realized by replacing with a kanamycin resistance gene, which subsequently was removed by I-SceI mediated double stranded break repair. In the study, crtI2 and upp were deleted by redαβ deletion system, crtI2, upp and cgp3 were deleted by recET deletion system which also can achieve point mutant in gene fasR for serine(AGT) to asparagines(AAT). The first step gene deletion mutant was successfully obtained, however, the second kanamycin resistance gene elimination step failed thus far, though many conditions were tried.