Part:BBa_K2992027
botR integration module for C. sporogenes with the lactose inducible system
Usage and Biology
This parts entry represents an integration module for the expression of botR at the pyrE locus of the C. sporogenes genome. This module comprises the botR gene of C. botulinum BBa_K2992002 under the regulatory control of the BgaRL lactose inducible system from C. perfringens wherein the divergent PbgaR BBa_K2992020 and PbgaL BBa_K2992023 promoter sequences, coupled with their native 5’-UTR and RBS sequences BBa_K2992022 and BBa_K2992024 respectively control the transcription of bgaR and botR. Transcription for bgaR is terminated by its native terminator sequence BBa_K2992021 whilst botR uses Tfdx derived from C. pasteurianum BBa_K2284012. In our project we use the transcriptional regulator of neurotoxin production from C. botulinum, BotR, to control the regulation of our volatile reporter operons and our fluorescent reporter FAST BBa_K2992000 through interaction with its own promoter sequence PbotrR BBa_k299012 and PntnH BBa_K2992001 whose genes are cognate members of the BotR regulon. Doing so allows us to use our surrogate host strain C. sporogenes as a model system for predicting botulinum neurotoxin production following food manufacture, through the detection of our chosen reporters.
Characterisation
We designed the Plac-botR construct to permit the inducible expression of our volatile reporters using lactose. Unfortunately, despite our best attempts, the construct was unstable when transformed into C. sporogenes thus preventing us from generating the desired genomic integrant.
However, we were still able to assess the feasibility of using the lactose inducible system from C. perfringens to modulate reporter gene expression in an inducible manner. We achieved this by constructing our Plac-FAST composite part BBa_K2992049 into pMTL 82151 and transforming this into C. sporogenes.
FAST production was indeed inducible using the PLac system. Reporter expression was predictable wherein only negligible activity was detected in the absence of induced whereas substantial activity could be detected following induction with 1-10mM lactose inducer. Taken together, these data demonstrate the feasibility of using an inducible system to regulate expression in our reporter strains.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 573
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 573
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 573
Illegal BglII site found at 633 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 573
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 573
- 1000COMPATIBLE WITH RFC[1000]
References
Cañadas, I., Groothuis, D., Zygouropoulou, M., Rodrigues, R. and Minton, N. (2019). RiboCas: A Universal CRISPR-Based Editing Tool for Clostridium. ACS Synthetic Biology, 8(6), pp.1379-1390
Dupuy, B. et al., 2006. Regulation of toxin and bacteriocin gene expression in Clostridium by interchangeable RNA polymerase sigma factors. Molecular Microbiology, 60(4), pp.1044–1057.
Hartman, A., Liu, H. and Melville, S. (2010). Construction and Characterization of a Lactose-Inducible Promoter System for Controlled Gene Expression in Clostridium perfringens. Applied and Environmental Microbiology, 77(2), pp.471-478.
Minton, N., Ehsaan, M., Humphreys, C., Little, G., Baker, J., Henstra, A., Liew, F., Kelly, M., Sheng, L., Schwarz, K. and Zhang, Y. (2016). A roadmap for gene system development in Clostridium. Anaerobe, 41, pp.104-112. 2019
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.
Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research, 31(13), pp.3406-3415.
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