Difference between revisions of "Team:Nottingham/Lab"

Ideas

Immediately after meeting for the first time, we were tasked with finding a project that we would be working on over the summer. At first, the idea seemed daunting but before we knew it, amazing (sometimes outrageous) ideas were being suggested.

Almost a month later, after numerous presentations, we narrowed down our potential projects to five:

Ideonella sakaiensis

Ideonella sakaiensisis a bacterium that can degrade plastic (PET) using two enzymes. It was discovered outside a plastic recycling facility in Japan and subsequently isolated by research teams at Kyoto Institute of Technology and Keio University(Yoshida et al., 2016). The idea of being able to tackle the plastic crisis affecting the oceans and marine life was a particularly appealing one. However, we quickly realised that we didn’t know how to transform the organism and the time-scale for how long it would take. Also, our project was very similar to other research being conducted and so it would be difficult to come up with a novel way to address the crisis.

Water bottle biosensor

According to the House of Commons Environmental Audit Committee, in their first report of the season (2017-19), in the UK alone we use ‘13 billion plastic bottles every year’ and ‘only 7.5 billion are recycled’. This prompted the team to suggest a biosensor that detects harmful substance in plastic bottles. As a result, the average consumer would buy fewer plastic bottles and reuse their bottle. However, many solutions immediately were proposed that counteracted the need for the biosensor-this included the fact that many people buy permanent water bottles that they wash and reuse; it would be more cost effective to campaign recycling efforts than to market a biosensor and it is cheaper to just recycle a plastic bottle and buy a new one than to buy a biosensor. Indeed in the report by the Environmental Audit Committee (potential stakeholders), they suggest increasing the number of water fountains in open spaces and improving recycling through a number of methods including Deposit Return Schemes (which incentivise consumers).

Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is a predatory bacterium that preys on other (Gram-negative) bacterial species (Rendulic et al., 2004). Its enzymes and their mechanisms are being studied in order to better understand the bacterium for future use as a possible therapeutic agent or as a method to penetrate biofilms (Kadouri and O'Toole, 2005; Sockett and Lambert, 2004). Our team was interested in engineering the bacterium to use it (and its enzymes) as a ‘pathogen eating machine’in food processes-for example targeting Clostridium botulinum, which releases the botulinum toxin. On top of it being an interesting species to work on, we had a leading expert on the Bdellovibrio species at our university. However, the main bacterium we were targeting (Clostridium botulinum) is a Gram-positive bacterium so is not recognised by Bdellovibrio. Also, it would take many weeks to engineer Bdellovibrio for single gene mutations, which is difficult to transform using random transposon mutagenesis. This was a project that required many years which our team did not have!

mRNA interference of Streptococcus mutans

Streptococcus mutansis one of a number of bacteria involved in tooth decay and is the most prevalent. According to Public Health England, in the UK, ‘almost a quarter (24.7%) of 5 year olds have tooth decay’ of which 3 or 4 teeth are affected. Dental health problems also have a heavy financial burden on the NHS-it spends around £3.4 billion per year on dental care. What makes S. mutans particularly hard to deal wit his its ability to form biofilms regulated by glucosyltransferases which catalyse sucrose to adhesive glucan. In particular, GtfB and GtfB seem to be the most important in biofilm production-mutations in the gtfB and gtfC genes disrupted microcolony formation on saliva coated surfaces(Koo et al., 2010). Our idea was to use a bacteriophage (a virus that only infects bacteria) to deliver micro RNAs or small interfering RNAs to silence those genes. One of our supervisors works with phage so she would be able to guide the wet lab team. We opted to silence the toxins rather than kill the bacteria because we wanted a way of preventing glucan formation without disturbing the balance of the oral microbiome

mRNA interference of Clostridium difficile

Clostridium difficile is an anaerobic bacterium capable of forming spores (meaning it persists in the environment). Clostridium difficil einfection(CDI) is a major infection which causes hospital and community acquired-diarrhoea. It particularly affects those who have long-term hospital stays (especially the elderly), are immunocompromised/immunodeficient (for example due to chemotherapy) and/or are on broad-spectrum antibiotics. CDI has a heavy financial burden-according to Zhang et al. (2017), the annual costs due to C. difficile infections in the US alone are an estimated $6.3 billion with almost 2.4 million days spent in hospitals. According to the Centers for Disease Control and Prevention, between 1999 and 2007 there was an increase in the estimated number of deaths due to CDI from 3,000 to 14,000 which was seen across Europe and Canada as well (McDonaldet al., 2012; Lessa et al., 2012). This has been linked to a hypervirulent, resistance strain of C. difficile.

Within the SBRC, our supervisors work with a wide range of Clostridial species in the Clostridia Research Group. Unlike with S. mutans, there was more experience within the team and so the supervisors would be able to work with us more closely and advise us on this project.

It was hard to decide between working with Ideonella sakaiensis, Streptococcus mutansor and C. difficile. But in the end, the team voted and chose mRNA interference of C. difficile which became our project-ClostridiumdTox.

Project design

C. difficile & phage characterisation

C. difficile strain SBRC 078 was isolated previously in the SBRC from clinical faecal samples and belongs to the hypervirulent PCR ribotype 078. The strain contains the genes tcdA and tcdB encoding for both toxins. Phage phiSBRC was previously isolated in the SBRC from an environmental sample and can infect and form plaques on C. difficile SBRC 078. A lysogenic version of C. difficile SBRC 078, which contains phage phiSBRC integrated into the bacterial chromosome, was created previously in the SBRC.

C. difficile growth analysis

The growth profile of the wildtype version of C. difficile SBRC 078 was compared to the growth profile of the lysogenic version of this strain. To assess this the growth of both strains was monitored for 24 hours and the OD at 600 nm was measured and the maximum growth rate was calculated using the equation

where t1 is the OD at the start of exponential phase and t2 is the OD at the end of exponential phase. This data was used to inform the model parameters and was required to ensure that in the human gut the lysogenic bacterial strains created in this project would grow in the same manner as the wild-type cells and therefore would outcompete them.

Phage burst size

Phage burst size was assessed to determine the number of infectious phage particles produced per bacterial cell during one infection cycle. This was determined by measuring the number of infectious phage particles (in plaque forming units per ml) produced over a time-course after infection of C. difficile SBRC 078 with phiSBRC. The titre of free phage at each time point was determined by enumeration of plaques using a plaque assay. C. difficile SBRC 078 was infected to a multiplicity of infection (MOI) of 1. The burst size was calculated as the Final Phage Titre/(Infection Phage Titre – Titre of Unbound Phages). This data was used to inform the model parameters.

Promoter library

Our project aims to supress toxin production in C. difficile and we chose two different strategies to pursue this aim. Briefly, these strategies involve either dead-Cas9 (dCas9) or antisense RNA (asRNA) to inhibit toxin production. Both strategies will require a careful consideration of the genetic parts involved in the device. Of particular importance is the choice of promoter employed to control expression of the dCas9, guide RNA or antisense RNA. Tailoring expression to an appropriate level is often an important design consideration in genetic engineering. In our case it was thought that the use of a strong promoter would be of greatest benefit for both strategies we pursued in order to maximise either the amount of guide RNA and dCas9 or the amount asRNA. Concentration of these components within the cell was expected to correlate with the degree of toxin suppression and since the objective was to supress toxin to the greatest extent possible, we aimed to find and characterise strong promoters within C. difficile. The promoters we chose to characterise were as follows:

All seven promoters were intended to be assessed in both E. coli and C. difficile. PCsp_fdx and PCac_thl were chosen since they have been used extensively in studies on C. difficile as well as related organisms and both are considered to be strong promoters (Heap, 2018; Heap, Pennington, Cartman, & Minton, 2009). A comparison of the two suspected strong promoters was made with the native promoters controlling toxin expression in C. difficile PCdi_TcdA and PCdi¬_TcdB. It was thought to be interesting and potentially useful to discover the strength of the toxin promoters and potentially their variance in their expression in different conditions. Three existing iGEM registry promoters were also chosen to be assessed in C. difficile. This served two functions, firstly it improved the registry in terms of part characterisation as there is currently no data on their use in Gram-positive organisms. Secondly, since these promoters have been well documented in E. coli they could give a good indication of the strength of the clostridial promoters when used in E. coli.

Occasionally cloning dCas9 in E. coli can be problematic, potentially due to unwanted off-target effects of the protein, the DNA binding nature of the enzyme or due to the size of the gene itself. To facilitate cloning and yet maximise dCas9 activity in C. difficile the ideal promoter would have low expression in E. coli,/em> and yet high expression in C. difficile. The choice of promoters and decision to assay them in both E. coli and C. difficile was designed to help us choose the optimal promoters for the toxin suppression projects, characterise existing iGEM registry parts in novel contexts and add to the registry potentially valuable clostridial/Gram-positive promoters. Two different assays were chosen to assess the promoters described above. In E. coli, a GFP assay was chosen due to its widespread use, ease, cost, precision and reliability. However, GFP assays have not been successfully used in clostridia and as such other reporter assays are commonly used. One such reporter assay is the GusA assay in which the expression of the reporter gene gusA can be accurately measured via the eventual release of a fluorescent compound 4-methylumberlliferone (4-MU). The assay relies on the fact that the protein encoded by gusA is a glucuronidase which converts the non-fluorescent 4-methylumberlliferyl glucuronide (4-MUG) into the fluorescent (4-MU).