Team:Nottingham/Project
Project description
What is ClostridiumdTOX?
Clostridium dTOX is the University of Nottingham 2018 iGEM project that aims to engineer a lysogenic bacteriophage with genetic constructs that will suppress the toxin production in Clostridiumdifficile, an anaerobic bacterium that causes hospital-and community-acquired diarrhoea.We will be using two different strategies to target C. difficile toxins: an antisense RNA system capable of inhibiting translation of toxin transcripts, and a dead Cas9 mechanism to inhibit transcription of the toxin genes.Each method will use different genetic constructs which perform different roles to ultimately decrease toxin production. In the climate of antibiotic resistance, our goal is to produce a specific, novel phage therapy that not only will reduce C. difficile virulence but will be easy to administer and is more affordable than current treatments.For more information on why we chose to tackle C. difficile infection, please see our abstract.For more information on phage therapy, please visit our ‘Human Practices Gold’ page.For more information on the impact of C. difficile infection and how our project will influence society, please visit our ‘Human Practices Silver’ page.
Antisense RNA
The use of antisense RNAis demonstrated in both pro-and eukaryotes but it isbest documented inplants.Post-transcriptional gene silencing is an epigenetic processutilised by plants,fungi and animalstotarget cellular and viral RNA.Plants target viral RNA with antisense RNA as an immunological defence mechanism against viral infection (Hamilton and Baulcombe, 1999).Upon encounteringviral RNA, the complementary antisense RNA strand binds it,forming a double stranded RNA molecule whichspeeds up the (RNA) degradation process and inhibits ribosomal binding (hence the initiation of translation).As a result, RNAinterference has been exploited as a technology for identifying gene function through inhibition of gene expression(Vaucheret,Béclinand Fagard, 2001; Hammond,CaudyandHannon,2001).For more information on why we chose antisense RNA specifically and on how we plan to use it, please visit our‘Antisense RNA’ page.
More informationDead Cas9
Discovered as a bacterial defence mechanism against viral infection, theCRISPR-Cas system has revolutionised the field of synthetic biology and made gene editing cheaper, fasterand easier(Schmidtand Platt, 2017; Sander andJoung,2014).In particular, a modifiedCas9 protein (dead Cas9), which has an inactive endonuclease and so is not catalytically active, has allowed for the repression of gene expression.Dead Cas9has been shown to interfere with DNA transcription and, when coupled with a guide RNA, is proven to be specific to its gene targets (Qiet al.,2013). However, unlike antisense RNA, the inhibition occurs pre-transcriptionally i.e. it interacts directly with the DNA rather than with the RNA. Dead Cas9 can be used to targetthe promoter region of a gene of interest using a guide RNA. Binding of the dead Cas9proteinto the promoter region hindersthe binding of the RNA polymerase thus inhibiting the initiation of transcription.
More informationAbstract
Antibiotics serve a critical role in remedying bacterial infections, however a major disadvantage to their use is the non-specificity of broad spectrum antibiotics that drastically kills off beneficial bacteria reducing the diversity of the gut flora. The use of antibiotics allows opportunistic pathogens like Clostridium difficile to take advantage of the dysbiosis caused.
A consequence of antibiotic misuse and the capability of bacteria to readily adapt to versatile conditions, has allowed antibiotic resistance in bacteria to become a major dilemma. Each year in the United States alone 2 million people are subject to infection from antibiotic resistant bacteria. Phage therapy is an alternative to antibiotics. The goal of our project was to engineer a bacteriophage which will infect C. difficile and express genetic constructs designed to suppress toxin production. We will pursue two strategies to achieve this; asRNA and dCAS-9, both of which will target the toxin genes tcdB and tcdA. Ultimately, we aim to produce a phage therapy which will reduce toxigenicity of resident strains of C. difficile without significantly affecting the native gastrointestinal microbiota.
