Team:Groningen/Template/MODULE/projects/parts/toolbox

We started with a toolbox that will make it possible to use Lactococcus lactis as a chassis. This toolbox contains a vector, a set of constitutive promoters, several genes from the nisin operon and an excellent fluorescent marker.

To get BioBricks in L. lactis, a compatible backbone is needed. For this, we used a well known plasmid that is known to work in L. lactis, pIL253, and made this BioBrick compatible. The plasmid is a shuttle vector that can be used in L. lactis and Bacillus subtilis. It carries erythromycin resistance as a selection marker and has a mRFP coding device between the BioBrick prefix and suffix, so selection on inserts in this vector is possible.

For expressing genes in L. lactis, promoters are needed. Together with the standard RBS (BBa_B0034) and the standard double terminator (BBa_B0015) they get genes transcribed and translated. The set of promoters that is used for the L. lactis toolbox is the promoter collection that was BioBricked by the Uppsala iGEM team in 2013. The promoters originate from L. lactis and have first been described by Jensen and Hammer in 1998.¹ The promoters were placed in front of a fluorescent marker in L. lactis, so their relative strength is known.

An important part of our project, that will also form a nice addition to the toolbox, is the nisin operon. The nisin operon is responsible for making and secreting the lantibiotic nisin in certain L. lactis species. Nisin inhibits the growth of a broad range of Gram positive bacteria, of which many are spoilage bacteria or pathogens. Nisin is therefore extensively used in the food industry as a preservative. Nisin forms pores in the membrane of the bacteria it kills and inhibits the peptidoglycan synthesis.²

The operon consists of the genes NisA, NisB, NisT, NisC, NisI, NisP, NisR, NisK, NisF, NisE and NisG and their related promoters, see figure 1. NisA is the gene that codes for the nisin precursor (1), that will be modified and transported out of the cell. The nisin precursor its serines and threonines are dehydrated by NisB and then the precursor is cyclized by NisC. After this process, the precursor is transported out of the cell (2). Here, the lead peptide is cut off by NisP (3).
Additional genes of the nisin operon are NisR and NisK (4). NisK is a membrane receptor that activates NisR when it binds nisin. NisR can then activate nisin inducible promoters, like PNisA and PNisI. At last, there are the nisin immunity genes, NisI, NisF, NisE and NisG (5). NisI, a lipoprotein, serves as the first defense, and is therefore always present in the cell that has nisin immunity. NisF, NisE and NisG form an ABC-exporter together. They provide extra immunity and become present in the cell when nisin is encountered in the environment.³

Of the nisin operon, NisA (K1365000), NisC (K1365003), NisR, NisK (K1365006) and PNisA (BBa_K1365008) were sent to iGEM HQ. NisA can be used in L. lactis NZ9800, a NisA deficient strain, to let it produce nisin.⁴ PNisA is a promoter that is induced by nisin. NisR and NisK can be used to detect nisin and activate nisin inducible promoters.

Last but not least, a fluorescent marker that has a high performance in L. lactis was BioBricked and added to the toolbox. This marker is a version of the sfGFP protein, called sfGFP(Bs), that was optimized for Bacillus subtilis. When the optimized sfGFP was tested, it showed high performance in L. lactis.⁵ The sfGFP(Bs) can be used for the testing of constructs, quantification of promoters or it can be coupled to other proteins to track them inside or outside the cell.