Difference between revisions of "Part:BBa K2992039"

Latest revision as of 14:51, 21 October 2019

gusA reporter construct with PntnH 5-UTR+RBS

Usage and Biology

This parts entry represents a gusA reporter construct under the regulatory control of promoter for the non-toxic non-haemagglutinin ntnH gene of C. botulinum. The construct comprises PntnH BBa_K2992001 and its associated 5’-UTR and RBS driving the expression of the reporter gene gusA from E. coli BBa_K2300032 with a strong clostridial terminator TFad from C. pasteurianum BBa_K2284012. gusA encodes a B-glucorinidase from E. coli which can be used in a histochemical reaction to quantitatively measure promoter strength. This is accomplished through the addition of the fluorescent substrate 4 methylumbelliferyl glucuronide (4-MUG) which undergoes a colorimetric reaction with GusA, proportionate to its intracellular concentration. The nature of the Gus assay permits the measurement of promoter strength directly from cell debris without the requirement for extended cell lysis procedures. Doing so allows for the detection of reporter gene-products from anaerobic organisms which have recently been removed from the anaerobic cabinet or which have been frozen directly thereafter. In our project we use gusA as an established reporter gene for anaerobic organisms to validate the promoters chosen for our botR and acetone production constructs.

Characterisation

The GusA reporter system was used to assess the promoter activity of our promoters chosen to drive the expression of botR from the genome of C. sporogenes and the expression of plasmid-borne acetone production pathways. The three composite parts, promoterless gusA construct BBa_K2992037, gusA reporter construct with Pfdx 5-UTR+RBS BBa_K2992038 and gusA reporter construct with P-ntnH 5-UTR+RBS BBa_K2992039 were assembled onto pMTL82121 plasmids and transformed into wild-type or promoter-botR genome integrants of C. sporogenes. GusA activity was then measured from the resultant cell lysates.

Pfdx provided considerable reporter activity in wild-type C. sporogenes (purple lines). As expected, PntnH was incapable of generating sufficient reporter without the presence of genomic botR (green lines). In the presence of botR, the construct generated detectable GusA (red lines) thus validating the positive transcriptional activation of Pntnh by the gene product of botR. Collectively these data indicate sufficient activity of PbotR and PntnH whilst demonstrating 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. Sequence and Features

Sequence and Features

References

Heap, J., Pennington, O., Cartman, S. and Minton, N. (2009). A modular system for Clostridium shuttle plasmids. Journal of Microbiological Methods, 78(1), pp.79-85.

Joseph, R.C., Kim, N.M. & Sandoval, N.R., 2018. Recent Developments of the Synthetic Biology Toolkit for Clostridium. Frontiers In Microbiology, 9, p.154.