Part:BBa_K4853000

Cyclase for nisin.

Lactococcus lactis nisC encodes an enzyme that catalyzes the creation of thioether bonds between dehydroamino acids and cysteine thiol groups, which are required for nisin to be modified¹

Usage and Biology

Figure 1. pACYCDuet-1-nisC restriction map. This map indicates all the possible restriction sites in pACYCDuet-1-nisC. The total construct is 5211 bp and nisC is 1274 bp.

Figure 2. Restriction digest to verify insertion of nisC into pACYCDuet-1. Beside a 1kb + ladder, the first lane contains a negative control, in which pACYCDuet-1 digested with Ssp1. The second lane contains pACYCDuet-1-nisC digested with Ssp1 with a band at 1274 bp, verifying that nisC is contained in the plasmid. Gel electrophoresis was conducted to separate the restriction digest products in a 1% agarose-TAE gel. The red asterisk highlights the nisC gene at 1274 bp.

A restriction digest was conducted to verify that our plasmid, pACYCDuet-1, contained our gene of interest, nisC. A band was observed at the expected length, 1274 bp, indicating we were able to successfully clone a gene of the expected length into our plasmid (Figure 2). We then confirmed that the DNA cloned into the pACYCDuet-1 was in fact nisC via Sanger sequencing.

Figure 3. Coomassie blue-stained 13% Tris-glycine SDS-PAGE analysis of NisC. Lane 1 contains whole cell lysate of E. coli BL21(DE3) cells containing plasmid pACYCDuet-1-nisC incubated with 0.4 mM IPTG at 37°C for 2 hours. Lane 2 contains whole cell lysate of E. coli BL21(DE3) cells containing plasmid pACYCDuet-1-nisC without IPTG induction. Note the presence of a band at 47.8 kDa, indicated by a red asterisk. The third lane contains whole cell lysate of E. coli BL21(DE3) cells with no plasmid, incubated with 0.4 mM IPTG at 37°C for 2 hours. Lane four has whole cell lysate of E. coli BL21(DE3) cells with no plasmid without IPTG induction.

Expression of a protein of the predicted molecular weight in E. coli BL21(DE3) was confirmed with an SDS-PAGE gel (Figure 3).

References

¹Plat, A., Kluskens, L. D., Kuipers, A., Rink, R., and Moll, G. N. (2011) Requirements of the engineered leader peptide of Nisin for inducing modification, export, and Cleavage. Applied and Environmental Microbiology 77, 604–611. Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1265
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]