Difference between revisions of "Team:TMMU China"

Establishment of bacterial or eukaryotic chassis for plugging-in and plugging-out genetic circuits and new-to-nature functionalities is a foundational work in synthetic biology. Lactic acid bacteria (LAB) are a group of generally recognized as safe(GRAS) bacteria and can be optimized to be the next generation of synthetic biology chassis, especially in the food industry and therapeutics. Among LAB,Lactococcus lactis is the most promising, but few 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. Besides, the currently used genome integration system is also time-consuming and labor intensive.

Therefore, we established a visual selection system by inserting a PnisZ promoter controlled lacZ gene into the His locus of L. lactis strain NZ9000 chromosome. Then the constructed strain would form blue colonies in the agar plate containing X-gal and nisin inducer. Further, lacZgene was replaced by the device of interest and the recombinant strains would form white colonies. Based on this strategy, we can easily pick up the white colonies among blue colonies. It is amazing is that the white colonies are exactly the desired recombinant strains because the lacZ gene has been replaced by the device of interest, while the blue colonies are not.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. So we’re proud to announce that “Mr. White“ is the ”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 the genome of L. lactis. We verified that the shortest mCherry device and the longest polysaccharide Vi device (14 kb) were successfully expressed and they were proved functional. To increase the expression efficiency in L. lactis, we optimized the gene expression system through introducing nisin immunity gene or nisin resistance gene into L. lactis so that it could tolerate a higher nisin concentration. To make proteins function at different locations, we also introduced signal peptide for secretion and cell wall anchoring domain for surface display into the plasmids,so as a result, proteins could be expressed intracellularly and extracellularly and also anchored to the surface. In this way, L. lactis might be able to become a suitable chassis with versatile equipments that can be widely applied in synthetic biology.