[Construction and identification of exogenous gene expression system in Lactococcus lactis].
OBJECTIVE
To construct and characterize an exogenous gene expression system in Lactococcus lactis.


METHODS
PCR-based gene assembly was used to synthesize the gene sequence containing P59 promoter, USP45 signal peptide, ribosome binding site and multiple cloning site. Plasmid pBS-pu was obtained after ligation of the assembled sequence with pBluescript II SK (+), and the terminator of USP45 protein was added to construct the recombinant plasmid pNBC1000. The gene coding for Streptococcus pyogenes M6 protein was amplified and cloned into pNBC1000 to obtain the plasmid pNBC2000. To characterize the expression system, enhanced green fluorescent protein (EGFP) gene was amplified from the plasmid pEGFP-N1 and cloned into pNBC2000. Laser scanning confocal microscope was used to observe the bacteria containing pNBC2000, pNBC2000-EGFP or pBS-EGFP.


RESULTS
Green fluorescence was visualized in the bacteria containing pNBC2000-EGFP, but not in the bacteria containing pNBC2000 or pBS-EGFP.


CONCLUSIONS
The plasmids pNBC1000 and pNBC2000 containing P59 promoter, USP45 signal peptide, ribosome binding site and multiple cloning sites are successfully constructed, which are capable of expressing exogenous gene in Lactococcus lactis.
Yang
Xiao-qiang
XQ
Institute of Digestive Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. harderware@163.com
Peng
Chun-xiu
CX
Jiang
Bo
B
Xing
Rui
R
Zhang
Shao-rong
SR
Chen
Xue-qing
XQ
D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics*; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; C014689:streptococcal M protein; D049452:Green Fluorescent Proteins