Part:BBa_K4437004

Antimicrobial region of nisin Q

This part contains the coding sequence for the antimicrobial region of NisQ, a variant of nisin.

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.

Sequence and Features

This sequence contains only the antimicrobial region of the peptide nisin Q.

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 37
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

We used the NisQ sequence within 3 composite parts (BBa_K4437001 aka "GB1", BBa_K4437002 aka "GB2", BBa_K4437003) in order to express NisQ protein.

Figure 1. PCR amplification of pSB1A3 plasmid containing NisQ, using T7 promoter and terminator-specific primers, indicating successful cloning. Lane 2 contains our expected band size at 331 bp

Figure 2. Diagnostic gel for the Xpress expression vector(BBa_K3945014) containing NisQ (within a composite part, BBa_K4437003) miniprepped-samples (samples digested at 37^oC for 60 minutes with no heat inactivation because of BamHI, therefore we expected the samples to not be fully digested). Lanes 4, 5, 6, and 7 show our expected band sizes. Lane 8 contains a negative control.

Figure 3. SDS-PAGE analysis of GST-NusA-NisQ (BBa_K4437003) samples from BL21 (DE3) E. coli strain autoinduced, using a Coomassie-blue stain. Large bands in lanes 2, 4, 5, 6, and 9 at 91kDa correspond to our expected protein size.

Figure 4. His-tag purified SDS-PAGE of GST-NusA-NisQ (BBa_K4437003) samples samples from BL21 (DE3) E. coli strain autoinduced, using a Coomassie-blue stain. Bands in lanes 3, 4, and 5 at 91kDa correspond to our expected protein size. Samples labelled "W-1" indicate wash 1, samples labelled "E-1" indicate elution 1.

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.