Team:Cardiff Wales
Point-of-care diagnostic testing can significantly affect patient prognosis.
Laboratory-based tests for sexually transmitted infections require specialised infrastructure and equipment for optimal performance [1]. STI testing is often conducted in resource-constrained environments where such resources are unavailable, limiting the use of test results in disease management, potentially impacting on patient prognosis [2]. Point-of-care testing (POCT) is operated outside of laboratories, near the site of and at the time of patient admission [3]. POCT has the potential to significantly reduce the impact of STI under these circumstances [4].
Cas-Find a novel system for POCT utilising Escherichia coli CRISPR/Cas9.
Cardiff_Wales has developed a novel bioluminescence detection system for point-of-care diagnostic testing, termed Cas-Find. In this system a Streptococcus pyogenes dCas9 isoform codon optimised for E. coli is fused to the C- or N- terminal fragments of a thermostable pH-tolerant Photinus pyralis luciferase mutant. The coexpression of sgRNA constructs targets these chimeric proteins to adjacent sequences, resulting in the reconstitution of luciferase activity and bioluminescence in the presence of luciferin. This constitutes the positive signal for DNA detection. We aimed to undertake a proof-of-concept study of this system using sgRNA targeted to the E. coli 16S rRNA locus, describing both the activity of this system in vitro, and the optimum distance between sgRNA targets.
Cardiff_Wales aimed to improve the function of the Vibrio Fischeri LUX operon, initially submitted to the Standard Registry of Parts by Cambridge in 2010 (a project which we termed FUEL). We characterised the Stokes shift of LUX operon output in the prescence of the RFP mKeima in both coregulation and coexpression conditions under a pBAD promoter, with potential applications in physiological imaging and more complex circuitry.
We have additionally undertaken collaborative work with Oxford, WasU_StLouis and Saclay.
