Construction of An Expression Vector with Nisin Resistant Gene nisI As A Food Grade Selection Marker for Lactococcus Lactis

Abstract: Lactococcus lactis is a kind of GRAS(Generally Regard as Safe, GRAS) bacterium that is widely used in food fermentation and biomedicine industry. In recent years, it has become a research focus in molecular biology of lactic acid bacteria that exploring some genes with practical applications significance by cloning and expression in lactic acid bacteria and construction a food-grade selection marker in Lactococcus lactis expression system.Objective:The erythromycin resistance gene was to be knocked out, which is located in the E.coli-Lactococcus lactis shuttle plasmid pMG36e. Construction of a E.coli-L.lactis shuttle expression vector pMG36N by introducing Nisin resistance genes nisI as a food grade selection marker.Methods:(1) According to the sequence of Nisin resistant gene nisI reported by GenBank, the nisI fragment was amplified by polymerase chain reaction with pLEB590 as template.(2) After being sequenced, the amplicon was confirmed by Blast from NCBI. Then, the nisI was subcloned into the E.coli-L.lactis shuttle vector pMG36e, resulting in the plasmid pMG36e-NisI.(3) The recombinant strain MG1363/pMG36e-NisI was obtained when the plasmid pMG36e-NisI was transformed into L.lactis MG1363 competent cell by electroporation. The recombinant strain carrying pMG36e-NisI showed the same growth curve and genetic stability as L.lactis MG1363, when the medium contained 20IU Nisin/mL.(4) In order to construct a food-grade vector pMG36N, erythromycin resistance gene from the recombinant plasmid pMG36e-NisI was to be knocked out by redesigning the pair of specific primers.(5) In order to examine the expression condition of EGFP in MG1363, the enhanced green fluorescent protein (EGFP) gene was inserted into the expression plasmid pMG36e-NisI and pMG36N respectively.Results:(1) A cloning vector pMD19-NisI was obtained.(2) The nisI fragment was amplified by polymerase chain reaction with pLEB590 as template. After being sequenced, the amplicon was confirmed by Blast from NCBI and the match rate is 100%. Then, the nisI was subcloned into the E.coli-L.lactis shuttle vector pMG36e, resulting in the plasmid pMG36e-NisI. The recombinant plasmid pMG36e-NisI was transformed into L.lactis MG1363 competent cell by electroporation. When the medium contained 20 IU Nisin/mL, the recombinant strain carrying pMG36e-NisI showed the same genetic stability as L.lactis MG1363. As suggests that the nisI gene could be used as a selection marker for construction of a food grade expression vector.(3) The expression vector pMG36e-NisI-EGFP and pMG36N-EGFP were constructed. EGFP, which was located in the downstream of NisI, can be expressed actively with the promotion of the strong constitutive promoter P32 in MG1363/pMG36e-NisI-EGFP and MG1363/pMG36N-EGFP.
Key words: L.lactis; food-grade expression vector; Nisin resistance gene; selection marker; gene knock-out;

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