Difference between revisions of "Part:BBa K2992026"

Latest revision as of 21:01, 21 October 2019

botR integration module for C. sporogenes with native RBS for polar transcription

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 coupled with its native RBS BBa_K2992011 to encourage transcription from the promoter of the pyrD gene following integration onto the chromosome of C. sporogenes. A strong clostridial terminator was included to prevent polar transcription of pyrE and any downstream genes on the chromosome of C. sporogenes 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 (hyperlink to comp parts) 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

In order to ensure the suitability of PpyrKDE coupled with the native RBS of botR for driving the expression of BotR in our volatile reporter strain, we assessed GusA reporter production on pMTL-82121 plasmids bearing the Pntnh-gusA construct BBa_K2992039. In this assay, GusA production should be a consequence of reporter gene transcription following interaction of PntnH with the C. botulinum-derived sigma factor BotR.

On analysis of the data. Pfdx provided the greatest level of promoter activity. PbotR generated a medium level of reporter activity which was marginally better than the promoterless construct permitting polar transcription from PpyrKDE. Crucially, the PbotR construct was incapable of generated detectable reporter activity in the absence of genomic botR. Collectively these data indicate sufficient activity of PbotR and demonstrate the critical activation of PbotR by the sigma factor BotR. Taken together, we conclude that the botR integration constructs should be applicable for acetone production studies.

Finally, the botR integrants and their controls were assessed for their ability to induce acetone production in our volatile reporter strains. The acetone production pathway csp_Pfdx-5-UTR+RBS-ca_thl-cb_ctfAB-cp_TFdx BBa_K2992035 was chosen for this purpose. The construct contained on pMTL82151 was transformed into the various promoter-botR genome integrant and control strains.

The data demonstrated appreciable acetone production of >2nM concentration when using either the native PbotR promoter and associated 5’-UTR+RBS or the RBS only construct to permit polar transcription from PpyrKDE</i>. Considerable acetone production (4-6nM) was observed when using the constitutive clostridial promoter Pfdx. Crucially, acetone production was comparably scant when botR was absent from the genome of C. sporogenes and when no promoter was used to drive expression of the acetone production operon. These data provide experimental validation for the production of acetone in C. sporogenes as a model for Botulinum toxin prediction in foodstuffs. Sequence and Features

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.

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.

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