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; Clostridium dTox.

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 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).

GusA assays can be performed in E. coli as well as clostridia and so both GFP and GusA assays were used in E. coli. Our GFP assay was inspired by our interlab experience as we thought it would be useful to use the protocols and calibration curves we obtained from the study to standardise our data. This would help us give context to the strength of the promoters by comparing them to the interlab positive and negative controls, using the calibrations curves generated through our interlab study to ensure that the results would be reproducible by any other laboratory using different equipment.

Heap, J. T. (2018). Stringency of Synthetic Promoter Sequences in Clostridium Revealed and Circumvented by Tuning Promoter Library Mutation Rates. https://doi.org/10.1021/acssynbio.7b00398

Heap, J. T., Pennington, O. J., Cartman, S. T., & Minton, N. P. (2009). A modular system for Clostridium shuttle plasmids. Journal of Microbiological Methods, 78(1), 79–85. https://doi.org/10.1016/j.mimet.2009.05.004

CRISPRi

Antisense RNA

Antisense RNA (asRNA) is another means of reducing gene function. In this strategy a piece of RNA is transcribed which is complementary to the coding strand of a target gene in the reverse orientation, in other words it is the antisense to it. This has the effect of sequestering the coding mRNA in an RNA-RNA duplex which is unable to effectively bind the ribosome meaning protein synthesis of that gene is inhibited. The RNA-RNA duplex molecule may also be targeted by RNases specific to double stranded RNAs meaning the target mRNA is degraded faster due to the presence of asRNA.

Many studies have utilised an asRNA approach to genetic studies. Typically asRNA will not be capable of completely eliminating gene function since target mRNAs may not encounter the asRNA molecule within the cell before being translated. For this reason, asRNA has primarily been used when gene ‘knockdowns’ are desired rather than total gene knockouts. In knockdown strains the expression of the target gene is reduced rather than entirely eliminated. Non-model organisms which are less genetically accessible frequently do not have established methods for creating gene knockouts. In this case asRNA is often used to gain initial insights into gene function since all that is required for asRNA studies is knowledge of the target gene sequence, a plasmid capable of replication in the organism and a means of transformation.

Our project aims to create a bacteriophage which will supress toxin production in C. difficile once integrated into its genome. To do this we will genetically modify a bacteriophage known to infect strains of C. difficile named phiSBRC. The bacteriophage will be modified to include constructs to supress toxin production either via a dead-Cas9 approach or via asRNA. There were several design considerations when approaching this problem. Firstly the construct should be capable of significantly supressing toxin production, it was not known whether dead-Cas9 or asRNA would be superior in this respect. Secondly, the eventual genetic construct we choose should be sufficiently small that we can alter the bacteriophage genome without affecting its normal function adversely. One potential limitation was thought to be the total amount of DNA which the bacteriophage could package into its head. In this respect asRNA could have a significant advantage over a dead-Cas9 approach since the total size of the genetic construct can be much smaller. Antisense RNA constructs can simply consist of a promoter and a short asRNA region of less than 100bp while the cas9 gene alone is more than 4kb long. However, since it was not known which approach would produce more effective toxin suppression both approaches were pursued.

C. difficile has two well characterised toxins which cause epithelial cells to undergo apoptosis these are TcdA and TcdB. It was thought that each construct we create should aim to supress both of these toxins simultaneously since research has concluded that each can operate independently from the other (Kuehne et al., 2010). The general form of our constructs therefore is to have antisense RNA parts downstream of promoters with a transcriptional terminator between these promoter-asRNA pairs. It was thought to use two different promoters to as to avoid unwanted recombination events within our constructs. As discussed previously, the optimal promoter for expressing the asRNA parts was thought to be the strongest promoter possible. We were therefore looking for the two strongest promoters in C. difficile we could find. Our results from the promoter assays we performed indicate that PCsp_fdx¬ is the strongest promoter assayed in C. difficile. Unfortunately we were unable to clone the GusA reporter plasmid for PCac_thl which was the other clostridial promoter expected to give strong expression. However, the PCac_thl GFP reporter plasmid was created and assayed in E. coli where it gave the highest expression of any promoter tested including the positive control used in the iGEM interlab studies. This result was consistent in our laboratory as well as that of our two collaborating teams representing Imperial and Warwick Universities. Since P¬Cac_thl outperformed every other promoter in E. coli, is derived from a clostridial organism, and has been routinely used in overexpression studies in clostridial research it was thought likely that it would have given strong expression in the GusA assay in C. difficile had the plasmid been cloned. As such PCac_thl and PCsp_fdx were chosen as the two strong promoters from which to express our asRNA parts.

When choosing the length of the antisense RNA we consulted the scientific literature. There is some contradicting advice on this topic with E. coli asRNA parts seeming to be significantly shorter than those used in the few asRNA studies we found performed in clostridia. There are important design considerations here since there is a compromise to be made. Longer asRNA parts seem to generally give a greater degree of suppression but are also more likely to give unwanted off-target effects. This is probably because they can bind the target mRNA more tightly but are also more likely to have regions of short similarity with other non-target mRNAs within the cell. With this in mind we chose to try two different lengths of asRNA binding to the coding region of the target gene as well as the entire region upstream of the gene expected to include the ribosome binding site. ‘Construct One’ has a coding region binding region of 24bp, this is the length suggested by a recent review paper on this topic (Hoynes-O’Connor & Moon, 2016). ‘Construct Two’ has a coding region binding region of 50bp, this is much longer though still significantly shorter than the hundreds of base pairs previously used in clostridial studies (Desai & Papoutsakis, 1999; Fagan & Fairweather, 2011). Both of these constructs target both of the toxin genes we are interested in.

Construct One diagram

Construct two diagram

Desai, R. P., & Papoutsakis, E. T. (1999). Antisense RNA strategies for metabolic engineering of Clostridium acetobutylicum. Applied and Environmental Microbiology, 65(3), 936–945.

Fagan, R. P., & Fairweather, N. F. (2011). Clostridium difficile has two parallel and essential sec secretion systems. Journal of Biological Chemistry, 286(31), 27483–27493. https://doi.org/10.1074/jbc.M111.263889

Hoynes-O’Connor, A., & Moon, T. S. (2016). Development of Design Rules for Reliable Antisense RNA Behavior in E. coli. ACS Synthetic Biology, 5(12), 1441–1454. https://doi.org/10.1021/acssynbio.6b00036

Kuehne, S. A., Cartman, S. T., Heap, J. T., Kelly, M. L., Cockayne, A., & Minton, N. P. (2010). The role of toxin A and toxin B in Clostridium difficile infection. Nature, 467(7316), 711–713. https://doi.org/10.1038/nature09397

Results

C. difficile & phage characterisation

C. difficile growth analysis

The growth of wild-type C. difficile SBRC 078 was compared with the growth of the lysogenic version of this strain. By measuring the OD at 600 nm of the two bacterial cultures over a 24-hour period. It was determined that the lysogenic strain has a slightly longer lag phase than the wild-type strain (Figure 1) but both strains reached the same maximum OD. The maximum growth rates for the two strains were similar with wild-type reporting a maximum growth rate of 0.26 µ/h and the lysogen measuring 0.23 µ/h. The negative control (broth containing no bacteria) reported a maximum growth rate of 0.007 µ/h showing no contamination over the time-period. The similar growth rates of the wild-type strain and lysogen showed that when lysogens were created in the gut as part of the therapy, the lysogens would be able to maintain their population over time in the same way as wild-type strains, therefore ensuring they are able to compete for nutrients and act as a probiotic to reduce colonisation of incoming toxigenic C. difficile strains.

Phage burst size

The phage burst size was calculated to determine the number of infectious phage particles produced from one bacterial cell during one infection cycle. A culture of C. difficile SBRC 078 was infected with phiSBRC to a MOI of 1 (infection titre of phage of 1.38 x 106 pfu/ml) and incubated for 15 minutes to allow phages to adsorb. The culture was washed of any unbound phages and then incubated under anaerobic conditions for 80 minutes. The number of phages in the supernatant was monitored. Figure 2 shows the number of phages present at various intervals over the period. It was observed that between 65 and 70 minutes and then 75 and 80 minutes the phage titre seemed to plateau slightly, indicating the end of the first burst cycle. It was determined by plaque assay that the final phage titre at the end of the first burst cycle was 4.2 x 106 pfu/ml and the number of unbound phages after the 15-minute incubation was 1.05 x 104 pfu/ml. These values were used to calculate burst size which was determined as 33 phage particles per cell. The burst size was a useful parameter for the phage model and allowed the number of phages over time in the model to be more accurately determined.

Promoter library

CRISPRi

Antisense RNA

The ultimate objective was to incorporate the described asRNA system supressing two C. difficile toxins into the phiSBRC prophage of C. difficile. The edited prophage could then be prepared from a stock C. difficile culture and used as a phage therapy treatment on patients suffering from C. difficile infections. To first demonstrate the efficacy of the asRNA constructs at supressing toxin production the two constructs we created were cloned into a plasmid vector suitable for transforming C. difficile. The C. difficile cultures harbouring antisense RNA plasmids were compared to wild type C. difficile in terms of supernatant cytotoxicity using African green monkey kidney epithelial cells of the ‘Vero’ lineage. C. difficile cultures were monitored over five days in terms of optical density as a read-out for bacterial growth and samples were taken, centrifuged and the supernatant filter sterilised in preparation for the cytotoxicity assay.

Cell supernatants of C. difficile contain the two toxins of interest TcdA and TcdB which are capable of stimulating mammalian epithelial cells to undergo apoptosis. It was thought that the supernatants from cultures containing our two antisense RNA constructs would have a lower concentration of toxins and therefore produce lower cytotoxic effects on the vero cells. Vero cells were grown in a 96-well cell culture plate using Dulbecco’s modification of Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS). After a confluent monolayer of epithelial cells was formed the sterile C. difficile supernatant was applied and the cells incubated for 24 hours at 37°C with 5% CO2. After incubation the medium-supernatant solution was taken and added to the LDH master mix solution, incubated in the dark at room temperature for 30 minutes before the absorbance at 492 nm was measured. Absorbance at 492 nm is a readout for cell death due to the released lactate dehydrogenase from lysed cells reducing NAD+ to NADH/H+ which is then used to reduce a tetrazolium salt into formazan. The formazan dye produced gives an absorption maximum at 492 nm and since the concentration of formazan correlates with the amount of lactate dehydrogenase released by the cells it can be used as a measurement of cytotoxicity.

Our results show that the supernatant toxicity of wild type C. difficile appears to plateau at 48 hours with no further increases observed. This plateau effect is likely produced by the concentration of toxin in the supernatant overcoming a threshold whereby the assay is no longer sensitive to any increases in toxicity. Both antisense RNA construct containing cultures take a around 120 hours to reach this plateau of toxicity as their rate of toxin production is signficantly lower. The rate of toxin production was taken as the OD-normalised LDH assay 492 nm absorbance value divided by the number of hours that the sample had been growing. Using this formula the wild type culture exhibited a toxin production rate of 0.0506 arbitrary units whilst construct one and two produced 0.0102 and 0.0074 respectively. Comparing these rates reveals that the antisense RNA construct one reduces the toxin production rate by 79.8% and construct two reduces the toxin production rate by 85.3%.

Figure legend.

Figure legend.