Part:BBa_K4437001

NisQ with a N-terminus 6x His-tag (NisQ-His)

Usage and Biology

Derived from Lactococcus lactis, nisin is a food-safe, antimicrobial peptide (AMP) that targets a wide range of Gram-positive bacteria by binding to lipid II on the pathogens membrane, creating a pore, and causing cell death [1]. Literature suggests that nisin Q (NisQ) demonstrates greater antimicrobial and antioxidant activity against pathogens compared to other variants of nisin, such as NisA (BBa_K1365000) [2]. Nisin’s optimal pH stability is between 2 and 7 but can maintain its antibacterial activity up to a pH of 12, and can also retain its antimicrobial activity at temperatures of 120^oC [2]. Unlike other AMPs, nisin is non-toxic to Gram-negative bacteria, meaning that successful recombinant expression in E. coli can be achieved without an inhibitory protein.

Design

Nisin GB1 (BBa_K4437001) includes, a T7 promoter, a lac operon, RBS, TEV protease site, 6XHis-tag, two enterokinase cut sites, nisQ, and T7 terminator. The two enterokinase sites serves to make sure the 6XHis-tag is cleaved off and surely leaves us with nisQ. The lac operon functions to be able to use different protein induction media such as autoinduction medias when expression protein. The presence of the lac operon will allow the bacteria to switch from a glucose sugar source to a lactose sugar source. The rest if the elements are regulatory elements necessary to express our protein.

Characterization

We have successfully characterized lanmodulin’s REE binding capabilities. To do this we conducted a metal recovery assay where an initial sample of neodymium was incubated with lanmodulin. In this assay an initial known sample of neodymium was incubated with the protein. The protein fraction was then removed using centrifugal filtration and the filtrate was analyzed using the colorimetric Arsenazo assay.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 220
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Zhou H, Fang J, Tian Y, Lu XY. Mechanisms of nisin resistance in Gram-positive bacteria. Annals of microbiology. 2014 Jun;64(2):413-20.
2. Mai HT, Van Hau N, Nghia NH, Thao DT. Expression and Purification of Nisin in Escherichia coli. Int. J. Life. Sci. Scienti. Res. eISSN. 2018 Jul;2455(1716):1716.