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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

GFP assay in E. coli

Our first objective was to quantify promoter strength in E. coli. To do this we synthesised each promoter upstream of wild type GFP [BBa_E0040] with the biobrick prefix/suffix flanking the part. The terminal restriction enzymes for the prefix and suffix were then used to clone the part into an empty pMTL84151 vector. pMTL84151 contains the ColE1 origin of replication for use in Gram-negative organisms as well as a Gram-positive origin of replication so that it can be used as a shuttle plasmid between E. coli and C. difficile.

The method used for our GFP assay can be found in our Labfolder. Briefly, reporter plasmids were transformed into E. coli DH5α after which an overnight culture was grown. The overnight culture was used to inoculate test cultures which were normalised to the same starting optical density (OD). After six hours of growth at 37°C shaking at 220 RPM the optical density was measured and cell samples were taken. Fluorescence was calculated using excitation at 485 nm and measuring emission at 525 nm. This fluorescence value was then normalised to the optical density to give fluorescence per OD unit. The positive [BBa_I20270] and negative [BBa_R0040] controls used were the same as those used in the 2018 InterLab study.

The strongest promoter of our set was P_{Cac_thl} (0.3235µM) which was around three times stronger than the positive control [BBa_I20270]. The second strongest clostridial promoter we tested (P_{Csp_fdx}) was slightly less strong than the positive control. The remaining two clostridial promoter P_{Cdi_tcdA} and P_{Cdi_tcdB} only weakly expressed in E. coli, though both showed activity significantly higher than the negative control [BBa_R0040]. The three existing parts we characterised with a novel RBS showed a range of expression levels as expected [BBa_J23106, BBa_J23114, and BBa_J23119]. We conclude that these promoters are capatible with the novel RBS part [Bba_K2715009] we added to them.

GusA Assay in C. difficile

As we need to establish data for promoter strength in C. difficile as well as E. coli, we needed to find an alternative assay that would work for anaerobic organisms. The GFP protein requires oxygen in order to form properly and fluoresce meaning it is not suitable as a reporter gene in this context. The Β-Glucuronidase encoded by gusA functions as a very sensitive and specific reporter gene in anaerobic organisms. The seven plasmids used in the GFP assay were modified to replace the gfp gene with gusA using NdeI and SpeI. For six of the seven gusA reporter plasmids cloning was successful with sequencing of the gusA showing no mutations. However, every clone of P_{Cac_thl} –gusA exhibited mutations within gusA. This is likely because of the toxin impact of gusA on E. coli and the very high expression levels of P_{Cac_thl} as shown in the GFP assay.

The six successfully cloned gusA reporter plasmids were transformed into a conjugative strain of E. coli named Sexpress. Sexpress was then used to transfer the reporter plasmids into C. difficile where the gusA assay proceeded.

The gusA assay was performed in 96 well plates in a plate reader using a similar protocol to the interlab study, except that 16 hour cultures were used rather than 6 hour, and the cells were lysed using lysozyme and sonication. The substrate for the Β-Glucuronidase, 4-methylumbelliferyl glucuronide (4-MUG), was added to the cell lysate immediately before the assay, and the production of the fluorescent compound 4-methylumbelliferone (4-MU) was tracked over 30 minutes. The rate of change in absorbance over this time period was recorded by the plate reader, and the gradient of the linear portion of the curve was calculated in order to give an indication of the quantity of Β-Glucuronidase in each strain. The results of the gusA assay are shown in the graph below.

None of the three existing registry promoters (BBa_J23106, BBa_J23114, and BBa_J23119) showed any detectable level of expression. This is somewhat unsurprising given that they are native to the distantly related E. coli, though they do have a ribosome binding region from clostridia which was intended to allow them to function in C. difficile. Of the two toxin promoters native to C. difficile only P_{Cdi_tcdA} showed any detectable activity and this was approximately seven times lower than the activity of P_{Csp_fdx}. This could be due to the assay being performed in BHIS medium which contains compounds which suppress the promoter. A research paper suggests that these toxin promoters are subject to catabolite repression (Dupuy et al., 2011). The strongest promoter in C. difficile was clearly shown to be P_{Csp_fdx}.

Antunes, A., Martin-verstraete, I., & Dupuy, B. (2011). CcpA-mediated repression of Clostridium difficile toxin gene expression, 79(December 2010), 882–899. https://doi.org/10.1111/j.1365-2958.2010.07495.x

CRISPRi

Antisense RNA

The ultimate objective was to incorporate the described asRNA system suppressing 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 suppressing toxin production the two constructs we created were cloned into a plasmid vector suitable for transforming C. difficile. The C. difficile cultures harbouring asRNA 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 asRNA 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 asRNA construct containing cultures take around 120 hours to reach this plateau of toxicity as their rate of toxin production is significantly 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 asRNA 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.

Conclusion

Our project aimed to show that C. difficile toxin production could be reduced with genetic constructs which could then be incorporated into a phage which targets strains of C. difficile.

The first step toward this end was to characterise a recently discovered phage in terms of its infectivity parameters. Phage phiSBRC was demonstrated to infect the C. difficile SBRC 078 strain effectively with the plaque/burst size assay showing that 33 phage particles are released per C. difficile cell. This result was used as a parameter in our modelling work. Another important parameter needed for the model was the growth rate of C. difficile wild type compared with the C. difficile lysogen in which the phiSBRC phage has integrated into the C. difficile genome. The respective growth rates were calculated by tracking the growth of each culture. It was concluded that there was little difference in the growth rate between C. difficile and the lysogen.

Having demonstrated that phiSBRC would be a suitable phage for infecting toxic C. difficile we next wanted to design a genetic construct which would be capable of suppressing toxin production. The two approaches we considered for this were dCas9 and asRNA. Both of these approaches required the use of strong, constitutive promoters. For this reason the next step for us was to characterise a range of promoters for strength in C. difficile. Whilst achieving this goal we also decided it would be beneficial to attempt to improve the characterisation of existing registry parts by measuring their expression in a novel organism. C. difficile is a Gram-positive anaerobic organism with significant differences to the E. coli chassis for which existing characterisation was performed. The existing registry promoters BBa_J23114, BBa_J23106, and BBa_J23119 were characterised for expression strength using a GusA assay in C. difficile. A new registry part which represents the ribosome binding region from the thiolase gene of Clostridium acetobutylicum was added to these promoter regions allowing them to be characterised in the context of having a different RBS than previously. In addition, four promoters have been added to the iGEM registry from C. acetobutylicum (Pcac_thl) [Bba_K2715010], C. sporogenes (PCsp_fdx) [Bba_K2715011] and two from C. difficile (PCdi_TcdA) [Bba_K2715012] and (PCdi_TcdB) [Bba_K2715013].

The four novel registry parts were characterised alongside the existing registry promoters in a GFP assay in E. coli as well as in a GusA assay in C. difficile. The most remarkable conclusion from the E. coli GFP assay of these promoters is that both of the suspected strong C. difficile promoter PCsp_fdx and Pcac_thl were stronger than any of the three existing registry promoters we assayed; with Pcac_thl producing around three times the concentration of fluorescein (0.3235µM) as the positive control used in the InterLab studies (0.0958µM).

Our main objective in characterising these promoters was to find a suitable pair of strong promoters to use in our subsequent dCas9 or asRNA projects. For this the GusA assay within C. difficile was most relevant since this is the chassis in which these constructs would be acting. The C. difficile GusA assay clearly showed that none of the three existing registry promoters from E. coli had any detectable activity in C. difficile. By far the strongest promoter we were able to measure was PCsp_fdx which was around 7.5 times stronger than the next strongest promoter we found (PCdi_TcdA). We were unable to clone the strongest promoter from the E. coli GFP assay PCdi_thl into a GusA reporter plasmid. This is likely because of the toxicity of the gusA gene in E. coli and since we know that PCdi_thl is the strongest of our promoters in E. coli it is unsurprising that this was the most problematic plasmid to construct. As a result we did not measure the strength of PCdi_thl in C. difficile, but due to its measured strength in C. difficile as well as its widespread use for overexpression studies in Clostridia we decided to select it alongside PCsp_fdx as a promoter to use in the next stage of our project.

Two asRNA constructs were cloned named ‘asRNA Construct One’ [Bba_K2715007] and ‘asRNA Construct Two’ [Bba_K2715008]. Both of these constructs target both toxin genes TcdA and TcdB with asRNA parts of varying length with asRNA Construct Two having longer regions of homology with 50bp of coding region verses 24bp for asRNA Construct One. These constructs were designed with the promoter results in mind, selecting the suspected two strongest promoters in C. difficile. Both constructs were assessed in terms of their ability to reduce C. difficile culture supernatant cytotoxicity on mammalian ‘Vero’ cells. The rate of toxin production was decreased by 80% and 85% by asRNA Construct One and asRNA Construct Two respectively. The main conclusion to draw from this result is that an asRNA strategy is viable for reducing C. difficile strain toxicity. Another conclusion of note is that having a longer region of homology with the target gene does seem to impact on the effectiveness of suppression significantly since asRNA Construct Two has a 5% greater effect whilst having 26bp extra of homology per toxin gene.

The other approach to suppressing toxin production was via a nucleolytically inactive Cas9 (dCas9). Demonstration of this approach did not progress as far as with asRNA because the cloning stage of this project was more time-consuming. While asRNA demonstrated a C. difficile supernatant with reduced cytotoxicity, our dCas9 approach was only validated in E. coli. However, positive results were obtained and future work should continue to pursue this approach. Six guide RNAs were evaluated in terms of their ability to target dCas9 to the toxin promoter region for toxin A (PtcdA). PtcdA was placed in control of the reporter gene gusA allowing quantification of the effectiveness of each guide RNA. Out of the six guide RNAs tested guide RNA 6 displayed the most consistently promising results with significantly less Gus activity implying that this guide recruits dCas9 to the PtcdA promoter region most effectively. Therefore guide RNA 6 will be used in future work when the dCas9 approach is trialled within C. difficile for its ability to reduce toxin production.

Future Work

Since we have demonstrated the effectiveness of asRNA at reducing C. difficile toxicity in this project, the obvious next step is to integrate our toxin suppressing construct into phiSBRC. This will involve taking the C. difficile lysogen with phiSBRC integrated into the genome and modifying it in the same way as we would modify the C. difficile genome normally. A recent paper (Wang et al., 2018) has described genome modification of C. difficile using Cas9 as a counter-selection mechanism forcing the cell to undergo homologous recombination with the delivered knockout plasmid to escape the lethal effects of Cas9. The recombination event which allows the cell to avoid the lethal double stranded break caused by Cas9 is directed by homology arms delivered on the knockout plasmid allowing researchers to delete genomic regions or introduce novel DNA into the genome. With this approach in mind we designed the plasmid pSBRC_Cas9_PhageIntegration_holin. This plasmid contains asRNA Construct Two which reduced toxin production by 85%, between homology arms directed at a gene within the phiSBRC prophage. The phiSBRC gene we chose to target was a holin gene which is thought to be responsible for cell lysis. Without this gene the progeny phage particles will not be able to burst out of the bacterial cell. This gene was chosen because it is one of the few areas of the genome which we are confident in ascribing function to and that function is not required to prepare more of the modified phage. Even without the phage being able to exit the bacterial cell it can still be induced and replicate itself and from there we can artificially extract phage particles ready for re-infection or delivery as a therapeutic. The other reason the holin gene was chosen is because of concerns around the size of phage genome which can be successfully packaged. It may be that the phage has evolved to be at or near to the limit of DNA which it can package. In this case replacing the holin gene which is of a similar size to asRNA Construct Two would mean that this is no longer an issue.

After knocking out the holin gene whilst simultaneously introducing asRNA Construct Two we would have a lysogenic strain of C. difficile with the modified phiSBRC integrated within the C. difficile genome. The asRNA Construct Two should still be active within the genome, as it would be constitutively expressed, and have a similar toxin suppressing effect to that demonstrated on a replicative plasmid in our results section. The cytotoxicity assays performed earlier will have to be repeated with the modified phiSBRC prophage taking the place of the replicative vector to ensure that the toxin suppression effect remains. It may be the case that since the asRNA construct on the genome is at a lower copy number than on a replicative vector it no longer displays such powerful toxin suppressing effects.

Having verified that the modified phiSBRC prophage retains its impact on toxin suppression the next stage would be to generate a second modified phiSBRC prophage which does not remove the phage holin gene. This is necessary because the modelling results suggest that having the phage able to occasionally enter the lytic cycle would be beneficial when put into practice. Instead of targeting the holin gene a region of non-coding DNA would be found and targeted with different homology arms to those used previously in pSBRC_Cas9_PhageIntegration_holin. Once the new modified prophage is created it would be necessary to ensure that the phage retains its ability to infect C. difficile and undergo the lytic cycle. For this reason a plaque assay would be performed as previously with the wild type phiSBRC and any difference in phage parameters would be re-entered into the mathematical model.

After this research is complete we would have a C. difficile lysogen containing a modified prophage which has been demonstrated to suppress toxin. This lysogen could be used to generate pure infectious phage particles which could be used in phage therapy. The next factor to consider would be the means of delivery to patients. After consulting with experts and discussion groups as detailed in the human practices it was decided that a capsule would be the optimal delivery method. As such the final stage of research in future work would be optimisation of the encapsulation of phage particles ready for application to patients.

Wang, S. et al. 2018. “Genome Engineering of Clostridium Difficile Using the CRISPR-Cas9 System.” Clinical Microbiology and Infection. https://doi.org/10.1016/j.cmi.2018.03.026.

InterLab

The aim of the iGEM InterLab study is to work towards a more reliable and repeatable measurement system to make synthetic biology an engineering biology. All participating laboratories first calibrated their instruments by obtaining standard curves using sodium fluorescein which was provided in our kits. This allowed us to fix settings such as top/bottom optic, gain and type of plate used so that all conditions were the same for our GFP and CFU protocols.

(Blue cells were raw data measurements, gold cells were calculated) After the machine was calibrated fluorescence and absorbance of GFP in 8 different devices was measured using the standardised method provided by iGEM. The results gathered were suggestive of which device had the highest strength of gene expression. The data collection sheet provided converted our raw plate reader measurements into arbitrary fluorescein and abs600 values as well as calculating the µM fluorescein per OD as shown in the following tables.

What did we gain from our InterLab experience? The InterLab study has allowed us to contribute to making synthetic biology more accurate through providing a series of standardised procedures of calibration and measurement tests. It helped our project as we were then able to use our new calibrated programmes to compare our promoters GFP assay data with iGEM standard data.

Labfolder

Protocols

Lab book antisense RNA experiments

Lab book promoter experiments

Lab book dCas9 experiments