We are the inaugural iGEM team from the fair country of Wales, based at Cardiff University. We aim to establish a tradition of synthetic biology amongst the undergraduate students at Cardiff and more widely in our community.
We have had a fantastically varied summer of 'design, test, build', and thoroughly enjoyed interacting with the worldwide community of motivated synthetic biologists at the Giant Jamboree.
Please read on, we hope you enjoy the Wiki as much as we enjoyed designing and implementing our project.
The Challenge: 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 . 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 . Point-of-care testing (POCT) is operated outside of laboratories, near the site of and at the time of patient admission . POCT has the potential to significantly reduce the impact of STI under these circumstances .
The Solution: 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.
The design of the Cas-Find project was significantly influenced by our conversations with experts involved in many areas that are important to consider for this type of diagnostic tool. These included a diagnostic biologist, an ethicist, Cardiff University safety officers and a retired nurse who specialised in the treatment of STIs. These interactions are highlighted within our human practice pages.
In addition we 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 aimed to characterise the stokes shift of the LUXoperon light output in the prescence of the mKeima RFO-varient in both coregulation and coexpression experiments conditions, with potential applications in physiological imaging and more complex circuitry.
We have undertaken collaborative work with Oxford, testing the efficacy of copper chelation utilising fluorescence lifetime imaging microscopy (FLIM) and WashU, to whom we provided analysis of ATP synthesis by their PCK and PGK constructs.
Finally we have taken the opportunity to engage with the public in a variety of outreach activities where we have highlighted the general potential of synthetic biology as well as discussing the specifics of our own project.