Difference between revisions of "Part:BBa K2992025"

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botR integration module for C. sporogenes with native promoter, 5-UTR+RBS

Usage and Biology

This parts entry represents an integration module for the expression of botR from the pyrE locus of the C. sporogenes genome. This module comprises a strong clostridial terminator sequence Tfad to prevent polar transcription from pyrD BBa_K2992013 and the botR gene of C. botulinum BBa_K2992002, under the regulatory control of its native promoter PbotR BBa_K2992012 and its associated 5’-UTR which contains the RBS BBa_K2992014. An additional strong clostridial terminator was included to prevent polar transcription of pyrE and any downstream genes on the chromosome of C. sporogenesBBa_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 PbotR for driving the expression of BotR in our volatile reporter strain, we first assessed it’s promoter activity using the fluorescent reporter for anaerobic organisms, FAST BBa_K2992002. We compared promoter activities of our chosen promoters PbotR BBa_K2992012 and PntnH BBa_K2992001 alongside three constitutive clostridial promoters Pfdxc114tBBa_K2992016, Pfdxt114c BBa_ K2715011 and Pthl BBa_ K2715010 and the E. coli promoter J23106 BBa_ J23106. The plasmids were cloned upstream of the FAST reporter gene and ligated into pMTL82151 plasmids. FAST reporter assays were conducted on both E. coli and C. sporogenes lysates following transfer of genetic material thereto.

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 generates 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.

PbotR was next assessed using the GusA reporter assay following genomic integration of the various promoter-botR constructs at the pyrE locus. Promoter-gusA construct were transferred into C. sporogenes on pMTL-82121 plasmids.

In a similar fashion to the FAST assays, 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.

Finally the <i>botR integrants and their controls were assessed for their ability to induce acetone production in our volatile reporter strains. The chosen acetone production pathway comprised the thiolase gene thl BBa_K2992008 and acetoacetate decarboxylase adc gene BBa_M36585 coupled with the two units of the ctfAB complex BBa_M36581 and BBa_M36582 which are all derived from C. acetobutylicum under the control of PntnH</i> from C. botulinum</i> BBa_K2992001. This 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 et al., 2019 RiboCas – update Dupuy and Matamouros, 2006 update Minton et al., 2016 Road map – update Raffestin et al 2009 update