Part:BBa_K4853001
nisin dehydratase (1)
Lactococcus lactis nisB encodes an enzyme that dehydrates certain serines and threonines in the nisin precursor1.1 It is essential to producing functional nisin. This part includes flanking sequences for efficient oligonucleotide primer design.
Usage and Biology
Figure 1. pRSFDuet-1-nisB restriction map. This map indicates all the possible restriction sites in pRSFDuet-1-nisB. The total construct is 6757 bp and nisB is 2999 bp.
Figure 2. Gel electrophoresis of restriction digest products in a 1% gel agarose-TAE gel to verify insertion of nisB into pRSFDuet-1. The first lane contains a 1kb+ ladder. The remaining lanes contain pRSFDuet-1-nisB cut with AflII. This has one cut site in the vector, pRSFDuet-1, and one in the insert, nisB. The red asterisk in lane six indicates nisB gene.
A restriction digest was conducted to verify that our plasmid, pRSFDuet-1, contained our gene of interest, nisB. A DNA band was observed in lane 5 at the expected molecular weight, 2999 bp, confirming that nisB was successfully ligated into our vector. We then confirmed that the DNA cloned into the pRSFDuet-1 was in fact nisB via Sanger sequencing.
Figure 3. Coomassie blue-stained 8-18% Tris-glycine SDS-PAGE analysis of nisB. Lane 1 contains whole cell lysate of E. coli BL21(DE3) cells with no plasmid of interest, incubated with 0.4 mM IPTG at 37°C for 2 hours. Lane 2 contains whole cell lysate of E. coli BL21(DE3) cells with no plasmid of interest, not induced with IPTG. Lane 3 contains whole cell lysate of E. coli BL21(DE3) cells containing plasmid pRSFDuet-1-nisB incubated with 0.4 mM IPTG at 37°C for 2 hours. Lane 4 contains whole cell lysate of E. coli BL21(DE3) cells containing plasmid pRSFDuet-1-nisB not induced with IPTG. The band of interest is marked with a red asterisk.
A protein band was observed at the predicted molecular weight, 118 kDa, suggesting that NisB is being expressed by the E. coli BL21(DE3) containing pRSFDuet-1-nisB.
References
1 Plat, 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 2990
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 107
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