Construction Of Genomic Library Of Lactobacillus Rhamnosus LS-8

The hurdle technology used for low, micro-processed food preservation, the biological preservative is one of its key points, in which bacteriocins are widely studied as one kinds of preservatives. Lactic acid bacteria is generally recognized as safe microorganisms, a lot of lactic acid bacteria can produce one or more antibacterial substances, including proteins or polypeptides which are called bacteriocins synthesized by ribosome biogenesis. These bacteriocins are efficient, non-toxic, non-residual, no drug resistance, etc, which make themselves become a focal point in food bio-preservation research.This experiment is to construct a genomic DNA library and study the possible bacteriocin encoding genes that may existed in Lactobacillus rhamnosus LS-8 from China’s traditional fermented foods, we draw the following conclusions through experiments.1. Alternately treatment by acridine orange and high temperature culture was used to eliminate plasmid, in which it was shown that plasmids were eliminated at the 24 th generation and completely disappear at the 29 th generation. Additionally, the results showed that the inhibitory effect did not change significantly between the 29 th and the 1th, which indicated that the bacteriocin was coded by genes in the genome and was not plasmid-encoded.2. The genomic DNA of LS8 was extracted and digested with Sau3A1 and Bam H1, after which 250bp~1000bp DNA fragments were recovered and linked with dephosphorylated pBluescript SK vectorby T4 ligase. The recombinant plamids were transformed into Top10 and recombinants were screened by the blue-white screening and resistance of ampicillin, by which 1480 clones were selected and verified by PCR.3. 101 DNA fragments were sequenced and five possible bacteriocin encoding genes were found according to blast search on NCBI, after which these five genes were amplified by PCR.