The construction of L-lactic acid over-producing genetic engineering strain

Abstract

Object of study: Lactococus lactis; L-Lactic acid; plasmid PK18mob; plasmid PNZ8150 Mission: Construct the genetic engineering strain- Lactococus lactis to get the high-efficiency, high purity and low-cost production of L-lactate. Knockout gene D-lactate dehydrogenase (ldhA) in genome of Lactococus lactis, with suicide vector PK18mob, with the method of homologous recombination. Then transformation expression vector plasmid PNZ8150-ldh to the Lactococus lactis. Methods : methods of molecular genetics(PCR, overlab PCR, TA colone; Enzyme restriction, extraction and purification of DNA, ligation,tranfomation, electroporation; sequencing validation), microbiological(cultivaton of microorganisms, single positive colone selection of Kmr .and by reversed selection of sucrose medium. ) spectrophotometric.