Part:BBa_K1830001

PnisA

Nisin inducible promoter and ribosome binding site

Usage and Biology

This promoter requires complementation from the Lactococcal nisin operon genes (nisZBTCIPRKFEB). Lactococcus Lactis is inherently resistant to the antimicrobial peptide nisin. These two operons are responsible for both the immunity of L. Lactis and nisin production. NisA is in fact the gene coding for the nisin precursor and its promoter (PnisA) will express any gene under its control when induced by nisin.

Instructions for use

Grow L. Lactis strain to an OD of 0.3

Induce expression with 10ng nisin/mL

Incubate for 20 hours

Literature information from 2024 East Coast Biocrew

The nisin system: Lactococcus lactis is a homofermentative bacterium that produces only one single product (lactic acid) from sugar. Nisin is an antimicrobial peptide is 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 within 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 K1830001, and it can be induced by NisRK.

Host strains: To exploit this auto-induction mechanism for gene expression the genes for the nisin signal transduction system (NisK and NisR) were first isolated. Then after being isolated, they were inserted into the chromosome of a subspecies Lactococcus lactis known as cremoris MG1363 which is nisin negative. This resulted in the Lactococcus strain NZ9000 https://doi.org/10.1016/S0168-1656(98)00100-X. When a gene of interest is placed on a plasmid or on the modified chromosome in NZ9000 cells, expression of the gene can be induced by adding about 0.1-0.5 ng/ml of nisin. Depending on several factors such as the presence of target signals, the protein can be expressed in the cytoplasm, membrane or medium. The NZ900 strain is available from MoBiTec (https://static.igem.wiki/teams/5272/nz9000.pdf)

Plasmids: The NZ9000 strain and its plasmids together make up the NICE (NIsin-Controlled gene Expression) system for gene expression in Gram-positive bacteria https://static.igem.wiki/teams/5272/nice-expression-system-handbook.pdf). 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://www.nature.com/articles/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 the large-scale production such as for the production of vaccines and lysostaphin https://doi.org/10.1186/1475-2859-4-16.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]