Our contribution for this year was to provide information on the nisin promoter by adding information to the parts page for K1830001, the pNisA promoter: Part:BBa_K1830001. We used a nisin-based induction system, which is a system for induction of gene expression in Gram-positive bacteria, to express our genes. We had to learn a lot about the plasmids and host strains for this system, such as that we needed to use Lactococcus strain NZ9000 which has the NisR and NisK genes that are needed for induction of the NisA promoter. We also had a lot of difficulty with the E. coli host strain that was first used for cloning our genes before transferring them to Lactococcus since normal DH5 alpha strains are not acceptable host strains. Instead, strain MC1061 is preferred, and we described this and some likely reasons why so that other teams wouldn’t have the same confusion. In our contribution, we included lots of links to the literature as well as links to manufacturers where other teams could buy the strains and plasmids. Below is the information that we added to the parts page.
Nisin System
Lactococcus lactis is a homofermentative bacterium that produces only one single product, lactic acid, from sugar. Nisin is an antimicrobial peptide made by Lactococcus and which binds to lipid II, a cell wall precursor, and then forms stable pores with diameters of 2-2.5 nm in target cells (https://doi.org/10.1128/JB.186.10.3259-3261.2004). Due to its broad host spectrum, it's often used as a preservative in food. As a 57 amino acid peptide, nisin is synthesized in the ribosome before being moved across the cytoplasmic membrane by NisT and then processed by the protease NisP (https://doi.org/10.1093/femsre/fuad023). Nisin has a unique property where the protein induces its own expression. If nisin is grown in small amounts in a culture it can lead to an auto-induction system. The nisin NisRK system activates the operons nisABTC and nisIFEG to synthesize and export more nisin (https://doi.org/10.1128/AEM.02392-15). The nisA promoter is this part and can be induced by NisRK.
Plasmids
The NZ9000 strain and its plasmids together make up the NICE (Nisin-Controlled gene Expression) system for gene expression in Gram-positive bacteria. The plasmid that we decided to use for the system was pNZ8148 which is widely available and can be bought commercially on several websites such as MoBiTec, Lifescience Market, and Novopro. This plasmid has an origin of replication, chloramphenicol-resistance gene, replication proteins repA and repC, the pNisA nisin-inducible promoter, and a transcription terminator. When transforming this plasmid into E. coli it is important to use host strain MC1061, a low concentration of chloramphenicol (10 μg/ml), and to grow the cells at 30°C. It is unclear why MC1061 is necessary, but there may be two reasons: (1) lack of promoter repression in other strains of E. coli that can cause toxicity when the cloned genes are expressed. The NICE Expression Handbook suggests that if MC1061 cells can’t be used, then you can instead transform a ligation mixture directly into a Lactococcus lactis strain. (2) There is also instability in RecA- strains that can lead to reorganization of the plasmid that can particularly lead to the loss of the chloramphenicol resistance gene (https://doi.org/10.1128/jb.179.7.2440-2445.1997).
Inducing the promoter
With increasing amounts of nisin and using the β-glucuronidase gene as a reporter, a linear dose-response is seen (https://doi.org/10.1128/aem.62.10.3662-3667.1996). A standard curve has also been demonstrated which shows a positive correlation between nisin concentration and Lactococcus’s ability to kill bacteria (https://doi.org/10.4315/0362-028X-70.5.1267)
Advantages and Disadvantages
Protein production with the NICE gene expression system in L. lactis strongly depends on the medium composition, the fermentation parameters, and the amount of nisin added for induction. These factors must be carefully optimized as they can lead to a significant increase in the amount of the target protein produced. For example, nisin production significantly decreases with an increase in the pH of the medium, which needs to be considered (https://doi.org/10.1038/srep27973). The system has advantages over other gene expression systems such as E. coli (https://doi.org/10.1007/s00253-005-0107-6 and https://doi.org/10.1016/j.biotechadv.2005.11.001). The system works in many Gram-positive bacteria, including some that don’t have other inducible promoters (https://doi.org/10.1128/aem.64.8.2763-2769.1998). Lactococcus and nisin have already proven to be safe for human consumption (https://doi.org/10.1080/10408399409527650). The system also has low protease activity, does not sporulate, and only has one membrane. The nisin-controlled gene expression system has already been shown to be useful for large-scale production such as for the production of vaccines and lysostaphin (https://doi.org/10.1186/1475-2859-4-16).