Construction of the expression plasmids

Three genes ( gshF, metK and cwaA) were cloned to plasmid pNZ8148 to form three modules (GSH module, SAM module and Adhesion factor module). Each module contained Pnis promoter (containing RBS sequence), the coding gene and terminator. Then, GSH module and SAM module were combined together to construct plasmid pNZ-GM. GSH module, SAM module and adhesion factor module were combined together to construct the final plasmid pNZ-GMcA.

Figure. 1 Schematic diagram of construction of mulita-functional plasmids.

Design and integration of three different modules (gshF, met K and cwaA)

Two fragments were chosen in the constructed plasmid to validate the validity. Fragment 1 named gm containing gene gshF and metK was 3670 bp and fragment 2 named cA containing gene cwaA was 3079 bp. As shown in Figure. 2, all the picked colonies contains gene gshF, metK and cwaA, illustrating that the plasmid pNZ-GMcA was successfully constructed.

Figure. 2 Validation of plasmid pNZ-GMcA. M represented marker. gm-1 to gm-5 and cA-1 to cA-5 all represented the same five randomly picked colonies.

SDS-PAGE detection of protein GshF and MetK

In order to validate the availability of the modules expressed in the plasmid pNZ-GMcA. We carried out SDS-PAGE analysis of protein GshF and MetK. GshF protein is about 86kDa and MetK protein is about 43 kDa. As shown in Figure. 3, expected bands of GshF and MetK protein were observed on the gel. Recombinant L.lactis containing pNZ-GMcA induced with different nisin concentration showed higher expression of MetK protein than wild-type. These results illustrated the successfully expression of GSH module and SAM module in plasmid pNZ-GMcA.

Figure. 3 SDS PAGE validation of gene gshF and metK expression in L.lactis. M represented marker. WT represented L.lactis NZ9000. 1-3 represented L.lactis/pNZ-GMcA induced with 100, 50 and 20 ng/ml nisin.