Difference between revisions of "Part:BBa K2992001"

Revision as of 00:19, 22 October 2019

PntnH

Promoter region for the non-toxic non-haemagglutinin component of the neurotoxin gene cluster in Clostridum botulinum.

Usage and Biology

Pntnh is found upstream of the 5'-UTR and RBS of the non-toxic non-haemagglutinin component (ntnh) of the neurotoxin gene cluster in C. botulinum. This promoter was used to drive expression of of the toxin regulator botR and our chosen reporter genes of interest which have been placed under the control of the neurotoxin and neurotoxin-associated promoters. Doing so allows us to link volatile reporter production with neurotoxin production following food manufacturing processes.

Characterisation

This basic part was used for the assembly of our composite parts and characterised using 3 difference reporters: GusA (BBa_K2992039), FAST (BBa_K2992044) and acetone (BBa_K2992036 ). This part was used as a promoter which functions in both the Gram-negative E. coli and the Gram-positive Clostridium sporogenens. More information can be found on our Results Page.





Characterisation of this promoter against Pfdx, Pthl and PbotR using FAST fluorescent assay, showed Pntnh to be an effective promoter in E.coli and only slightly stronger than no promoter in C.sporogenes.
In the C. sporogenes experiments, adequate expression was detected for each of the clostridial promoters chosen for study. The two Pfdx derivatives generated the greatest level of reporter activity whilst the two C. botulinum promoters generated much lower levels of activity. Reporter activity appeared to be generally higher when analysed from the E. coli lysates as opposed to the C. sporogenes lysates. In those experiments, activity from the PbotR and Pntnh constructs were considerably greater than the no promoter control.

Sequence and Features

References

Raffestin, S., Dupuy, B., Marvaud, J. and Popoff, M. (2004). BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botulinum type A and Clostridium tetani. Molecular Microbiology, 55(1), pp.235-249.