Construction Of Genetical Manipulation System In Escherichia Coli With D-Alanine Auxotrophy As Seletion Marker

That the extensive use of antibiotics for human health and the development of society was contributed, but it was also increasingly clear that the antibiotics were harm to human with the continuous improvement of the people’s understanding of biological evolution and the scientific progress, which had attracted more and more people’s attention. Currently, vectors used in molecular biology research were all using the resistance gene as a selection marker. The main fuction of racemase in vivo was to genarate D-alanine from L-alanine, for the majority of the bacterical cell wall peptidoglycan layer synsthsis requries. The study showed that the deletion of racemae gene was lethal for the growth of mutants,and it did not contain D-type amino acid in the normal medium. So the racemase gene could be used as a selection marker in the genetic manipulation.In this experiment, both air and dadX encoding racamase from E. coli DH10B were deleted by using Red recombination system mediated gene-knockout technology. The mutant strain of both alr and dadX deleted was a D-alanine auxotroph.The final concertration of 50μmol·L-1 D-alanine in LB medium was the lowest to maintain growth, and 200 μmol·L-1 D-alanine allowed it to grow normally. The measurement results of growth curve showed that, compared with the wild strain DH10B, mutant could grow normally, but the cell concentration was slightly lower in the whole growing period. The value of OD600 measured was away from 0.5 approximately and the number of mutant colonies was about half of the wild type in logarithmic medium-term and stabilization period, but we did not find a significant defference by using microscopic to examination colony morpholog. The mutant could be complemented, indicting that the D-alanine could be used as a selection pressure.Based on the above theoretical and experimental results, we built a non-resistant vector pECO21 by the method of overlapping extension PCR. pECO21 Vecoter reserved the general characteristics of cloning vector, but the difference was that D-alanine was used as a selection marker in stead of resistance gene.Because of pECO21 having BamHl restriction site, it could not only be used for general cloning systems,but also constructing bacterial artificial chromosome(referred to BAC)library, reaching 90% transformation efficiency. Besides, alanine racemase gene could be used as a food-grade selection marker and racemase as well as played an great role in the treatment of bacterical diseases.