Part:BBa_K2992015

5-UTR containing RBS for ntnH gene from C. botulinum

5’-UTR containing RBS for ntnH gene from C. botulinum

Usage and Biology

The native 5’-UTR containing the RBS is found naturally upstream of PntnH (BBa_K2992001) in C. botulinum. ntnH encodes the non-toxic non-haemagglutinin component of the botulinum neurotoxin complexes. Its expression is directly regulated by BotR (BBa_K2992002). In our project we use the regulatory region of ntnH to drive expression of our chosen reporter genes of interest. Doing so, allowed us to link BotR expression with the transcriptional activation of our reporter genes.

Characterisation

This basic part was used for the assembly of our composite parts and characterised using 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 (comprising parts BBa_K2992001 and BBa_K2992015) against Pfdx, Pthl and PbotR using FAST fluorescent assay, showed that Pntnh is a medium-strength promoter in E.coli, while being very weak in C.sporogenes (only slightly stronger than the promoterless control), as expected in the absence of BotR.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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.