Difference between revisions of "Part:BBa K2992044"

Latest revision as of 22:53, 21 October 2019

FAST reporter construct with PntnH 5-UTR+RBS.

Usage and Biology

This parts entry represents a FAST reporter construct under the regulatory control of PntnH for the non-toxic non-haemagglutinin ntnH gene of C. botulinum. The construct comprises the strong clostridial promoter PntnH BBa_K2992001 and its associated 5’-UTR containing the RBS BBa_K2992015 driving the expression of the fluorescent reporter gene FAST BBa_K2992000. Transcriptional terminator occurs through the activity of the strong clostridial terminator TFdx BBa_K2284012. FAST is one of the few fluorescent reporters available for effective use in anaerobic organisms. FAST is derived from Halorhodospira halophila and has been codon optimised for fluorescence studies in the genus Clostridium. In our project we couple FAST with the natural promoters of the BotR regulon thus linking reporter fluorescence with botulinum neurotoxin production. In doing so, we hoped to generate our surrogate host strain as a model for predicting neurotoxin production in foodstuffs following food manufacturing processes.

Characterisation

In order to assess the feasibility of linking reporter production with Botulinum neurotoxin production, we first generated our botR-integrants of C. sporogenes. botR expression was either driven by the native PbotR promoter BBa_K2992025, or the botR RBS without an accompanying promoter to permit polar transcription from PpyrKDE BBa_K2992026. Thereafter, promoter-FAST constructs comprising PntnH BBa_K2992044, Pfdx BBa_K2992043, or the promoterless control BBa_K2992042, upstream of FAST, were cloned onto pMTL82151 vectors and transformed into the abovementioned reporter strains. We then assayed for the presence of FAST in C. sporogenes lysates over a 48h period.

Considerable FAST activity was detected when botR was expressed from the genome of C. sporogenes using its native promoter when coupled with plasmid-borne PntnH-FAST (red lines). Crucially, the level of FAST detected in the absence of genomic botR was comparable to the promoter-less FAST plasmid (green vs orange lines). Plasmid-borne Pfdx-FAST generated sufficient reporter activity across all time-points. These data demonstrate that genomic botR can be used to regulate reporter gene expression to appreciable levels when placed under the control of the PntnH promoter. The FAST data corroborates the data obtained from our acetone production experiments, thus consolidating the suitability of our C. sporogenes reporter strains as models for the prediction of Botulinum neurotoxin production.

Characterisation of FAST using different Clostridium and Escherichia coli promoters.

The part was characterized through a fluorescence assay in E. coli as well as in C. sporogenes, along other promoters to assess their relative strength. Fluorescence is reported as Molecule Equivalent Fluorescence per Particle (MEFL/particle) as per the recommendation of the iGEM measurement Hub.

This part was characterised using fluorescent assay, and compared against FAST expressing constructs with a range of other promotersm including: Pfdx BBa_K2992043, Pthl BBa_K2992045, PBotR and a promotorless FAST construct BBa_K2992042

The first observation from the expression of the FAST protein using different Clostridium and E. coli promoters is that FAST is a suitable reporter gene, both in E. coli and in Clostridium sporogenes. Indeed, quantifiable levels of fluorescence were recorded in between 6.3*10³ MEFL/particle and 1.1*10⁶ MEFL/particle. However, it is worth noting, the assembly of the strong promoter BBa_J23119with the FAST gene only produced colonies with heavily mutated BBa_J23119, indicating that FAST expression could be toxic under the control of such a strong promoter. It is however somewhat surprising that Pthl was not also mutated, as last year’s [http://2018.igem.org/Team:Nottingham/Collaborations#warwick-and-imperial interlab study] consistently found that Pthl was a stronger E coli promoter than PJ23119. .

Sequence and Features

References

Streett, H., Kalis, K. and Papoutsakis, E. (2019). A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Clostridium Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST). Applied and Environmental Microbiology, 85(14).

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.