The Studies On Plasmid Extraction Method From Lactic Acid Bacteria And Transformation Of Lactic Acid Bacteria By Electroporation

Most Lactic acid Bacteria(LAB)are commonly considered as safe probiotics, and they are also considered to be safe bacteria with a GRAS (generally regarded as safe) status. Researching of food grade LAB vector - receptor system and expression the foreign gene in LAB has become the research hot spot for the food-grade inducible gene expression system. In this paper, LAB were isolated from the fermented foods at frist, and then extracted the high-purity plasmids from LAB. Furthermore appropriate plasmid was selected, sequenced and analyzed. Finally, the electricity transformation method was used for the capability of Lactobacillus plasmids and host matching reserch.8 strains were isolated from pickle and yogurt. They were Gram-positive bacteria and identified to be LAB with morphological and physic-chemical analysis. They had no spore, and grew in condition of 5% of CO2. Homology comparison with gene sequence in genbank proved that six of them are Lactobacillus plantarun and two of them are Streptococcus thermophilus. They were 99% similarity respectively.By combining the extraction method of plasmid from E.coil and the method of plasmid from LAB reported before to improve the common methods used in plasmid extraction kit, this paper developed a fast, safe and high efficient method for extraction of plasmid DNA from lactic acid bacteria. Experimental results showed that, the developed method showed an increased extraction efficiency, as well as good reproducibility.The optimal concentration of lysozyme was proved to be 20mg/mL, the optimal processing time was 30min, and the usage toxic ethidium bromide was avoided during the whole process of extraction.pLPS1 was chosen as the research object, which were isolated from Lactobacillus plantarun.After pLPS1 sequence was detected, and analysis through bioinformatics methods showed that, the GC content of the gene was 38.03%.The open reading frame NO.1 was from 267 to 1448, encoding 393 amino acids, and the theoretical molecular weight of which was 44.3 kDa. The secondary structure of protein was composed of 44.27%%α-helix, 43.77% random coil, 7.38% extended strand and 4.58%β-turn. A hydrophobic region of about 20 amino acids as well as a signal peptide was detected in the N-terminal of the protein.Using the Lactobacillus plantarun CGMCC1.555 as the host, best electroporation efficiency was 1909 cfu/μg DNA obtained after the voltage is 2.25 kV, concentration of glycine is 2.95%(W/V), and 3.69 h culture time; Using the Lactococcus lactis ssp. Lactis CICC 6060 as the host, best electroporation efficiency was 1375 cfu/μg DNA obtained after the voltage is 2.38kV, concentration of glycine is 2.68%(W/V),and 6.19 h culture time.