Difference between revisions of "Part:BBa K2992038"

Revision as of 14:09, 21 October 2019

gusA reporter construct with Pfdx 5-UTR+RBS

Usage and Biology

This parts entry represents a GusA reporter construct under the regulatory control of the strong constitutive clostridial promoter Pfdx. The construct comprises Pfdx BBa_K2992016 and associated 5'UTR+RBS BBa_K2992017, the reporter gene gusA from E. coli BBa_K2300032 and the 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

Data incoming.

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.