Team:TMMU China
Abstract
Establishment of bacterial or eukaryotic chassis for plugging-in and plugging-out genetic circuits and new-to-nature functionalities is the foundational work for synthetic biology. Lactic acid bacteria (LAB) are a group of generally recognized as safe bacteria and can be optimized to be the next generation of synthetic biology chassis, especially in the food industry and therapeutics. Lactococcus lactis is the most promising LAB chassis, but little efforts have been made to improve it to be a better chassis. On the other hand, the device introduced into L. lactis by plasmid is unstable and the antibiotic resistant genes are forbidden to be used in food and therapeutics. The currently used genome integration system is also time-consuming and labor intensive.
Here, we established a visual selection system by insertion of a PnisZ promoter controlled lacZ gene into the His locus of L. lactis strain NZ9000 chromosome. The constructed strain forms blue colony in the agar plate containing X-gal and nisin inducer. To clone the device of interest, lacZgene was replaced by the device of interest and the recombinant strains forms white colonies. Based on this strategy, we can easily pick up the white colonies from the pool of blue colonies. The amazing thing is that the white colonies are the desired recombinant strains because the lacZ gene is replaced by the device of interest, while the blue colonies are not. This time we can easily find the right colonies by only picking up the white colonies. “Mr. White is Mr. Right!”
We further tested the efficiency and capacity of the markerless visual selection system for knocking devices into the genome of L. lactis. 5 devices with different lengths (ranging from 1 kb to 14 kb) were inserted into genome of L. lactis We verified the shortest mCherry device and the longest polysaccharide Vi device (14 kb) were successfully expressed and functional. To increase the expression efficiency in L. lactis, we optimized the gene expression system to tolerate a higher nisin concentration through introducing nisin immunity gene or nisin resistance gene into L. lactis. To make proteins function at the right place, we also introduced signal peptide for secretion and cell wall anchoring domain for surface display into versatile plasmids. By this way, L. lactis became the right chassis with versatile equipments that can be widely applied in synthetic biology.
