Team:Nottingham/Results

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Results

As the wet lab was not available to us due to the global COVID-19 pandemic, we therefore lack significant experimental results. Instead we will present the in silico results we have produced and what results we would expect if we were able to get into the wet lab!

Modelling Subgroup

The modelling subgroup was presented with the task to identify which pathway to produce DBHB would be most beneficial for our project. This was assigned to our structural modelling group who used a MATLAB based tool, CellNetAnalyzer [1], and COBRApy [2] to determine which pathway was superior. The two pathways are discussed in more detail on the Model and Design pages of our wiki. Using flux balance analysis (FBA) and elementary mode analysis (EMA) we arrived at the conclusion that DBHBA was the pathway that we needed. Compared to DBHBB, the pathway we have chosen provides a higher maximum theoretical yield of DBHB, a greater percentage of routes that form DBHB (84% compared to 67%), and has a higher productivity, for both DBHB and ATP.


With DBHBA concluded as the pathway of choice, this information was then passed onto our kinetic modelling group and the rest of the team. Using kinetic modelling we were able to determine an optimal administration strategy for our DBHB producing probiotic. We concluded that 48-hour dosage administration was feasible to produce an average DBHB level of 4.8 mmol. We had aimed for weekly administration as meetings with Dr. Morrant and Nicola Cook (document in our HP wiki section) suggested that it was often difficult at times to get patients suffering with neurodegenerative diseases to take medication. So the less they are required the better. Unfortunately, weekly administration was not feasible due to significant oscillations in the levels of DBHB.


Additionally, it was established that the level of DBHB could be regulated by the dosage of a growth controlling nutrient in the capsules.

As for the initial amount of spores, we found that:

  1. It determines how long it takes to approach a stable oscillating growth and product formation of the mutant.
  2. The final level of biomass and product is unaffected by the initial amount of spores.
  3. The system seems to approach a stable limit cycle.

This is a very desirable behaviour for our drug because it means that slight changes would not disrupt the effectiveness of the drug. This allows for expected variations such as the time a patient might take the medication, how they take it (e.g. before or after a meal), their diet and similar.

DBHB Subgroup

To achieve our aim of producing DBHB in C. sporogenes we needed to assemble a synthetic gene construct. Our first result was the identification and in silico construction of two pathways that theoretically should produce DBHB. The first of these ketone production operons (KPO) was called the acetoacetate pathway (or DBHBA) and the second named the Hydroxybutyrate-CoA Pathway (or DBHBB). These pathways can be found in more detail on our Design page.

We then designed shuttle vectors harbouring our two pathways to test which pathway would be more favourable (Figure 1). We would conjugate these into C. sporogenes, maintaining the plasmid by antibiotic selection and expect to find one producing more DBHB than the other, analysed by gas chromatography of culture supernatants. This result would then inform our pathway choice.

Figure 1. Vector maps to show an example of the shuttle vectors designed using our two synthetic KPOs. Both maps are of the KPO, DBHBA (left) and DBHBB (right), driven by the native fdx promotor. Equivalent vectors for both KPOs downstream of the Px (no promotor), Pntnh and Plac promotors were also designed (see the DBHB lab book). - Vectors were designed using SnapGene

Results from the modelling subgroup suggests that the DBHBA would be more favourable and likely to produce more DBHB. This result was also further confirmed as the tesB gene found in our DBHBB pathway also acts on the S-isomer of 3-hydroxybutyrate-CoA (3HB-CoA), and could therefore also result in S-3-hydroxybutanoate (S3-HB) and not the R-3-hydroxybutanoate (R3-HB or ‘DBHB’) isomer we desire. From a human practices (HP) meeting with Professor Clarke we learned that the S isomer could be detrimental to health and, thus, in the absence of experimental data we choose to pick the DBHBA pathway.

Once the DBHBA KPO was chosen, we proceeded to design plasmid vectors to integrate this pathway into the C. sporogenes chromosome to allow stable expression of the pathway. These vectors are based on a CRISPR/cas9 mediated genomic editing tool for clostridia called RiboCas [3] (Figure 2). After design, assembly and conjugation of the integration vectors into C. sporogenes , we would expect to see similar levels of DBHB in the gas chromatography analysis as seen in our shuttle vector harbouring strains.

Figure 2. An example vector map of the plasmid vectors used to integrate the DBHBA KPO into the C. sporogenes chromosome. These vectors were based on the RiboCas system and needed addition of the cargo (promotor and KPO), homology arms (in pink) and guide RNA (in blue). This map shows the vector that would be used on make the DBHBA KPO (in orange) under control of the Pfdx (in grey). Equivalent vectors to integrate the Pntnh and Plac variants were also designed (see DBHB lab book) - Vectors were designed using SnapGene.

We would additionally preform growth curves and spore assays on each of the integrated KPO strains to ensure addition of these genes (and removal of pyrE) would not affect growth and spore production, respectively. When preforming these assays alongside our wildtype (WT) C. sporogenes, in which no genetic manipulation has occurred, we would hope to see no significant difference between WT and the KPO integrated strains.

To give more control and temporal expression of DBHB production genes, we choose to use three different promotors upstream of the KPO in all of our vectors; the native constitutive Pfdx, inducible PPlac and PPntnh. We would expect to see the following results for DBHB production from each of these promotor variants if we were in the wet lab, shown in Figure 3 below.


Figure 3. Graphs created to show the expected DBHB production profiles of each strain dependent on the promotor variant upstream of the KPO. The fdx promtor should give us the highest levels of DBHB and should start production from t=0. The no promotor variant (Px) will act as a negative control with no DBHB produced across the time course. The ntnh promotor should give temporal express of DBHB, with production starting at 8 hrs and peaking at 48 hrs, as shown in the graph on the left. The lac promotor is inducible by addition of lactose to the culture, so once added (in the graph on the right after 24 hrs) the levels of DBHB should begin to increase and give us more control over the levels of DBHB.

Control Subgroup

The control subgroup was tasked with regulating the therapeutics ability to negatively affect the environment after its role has been complete. The mechanism we would use involves an anhydrotetracycline inducible promoter attached to various genes associated with successful sporulation. We had to adapt CRISPR techniques to our project, a pRECas1 editing vector was manipulated to suit each of the sporulation gene targets. For each of the gene targets, templates of this pRECas1 vector were made including the left and right homology arms as well as the aTc-inducible promoter cassette. These are the editing templates for the gene targets. Primers were designed to successfully amplify these sequences during PCR. The next task was to find protospacers for the Cas9 to cut the DNA at; increasing the ability of the template to integrate into the C. sporogenes genome and create guide RNA to direct this Cas9. This was completed for all the target genes. Future experiments would aim to create this triple mutant for the target genes.

Routes of Administration

The routes of administration subgroup focused on the formulation of NeuroTone: what it should look like and which ingredients it should contain. As we did not have access to the wet lab, all our decisions were made based on information found in the literature.


The first decision that we had to make was how to administer NeuroTone to the patients: as part of a drink (yogurt, for instance), as a sachet or as a capsule. After some research, we decided to use a capsule: the formulation is simpler (reduced risk of premature germination of the spores) and it is more suitable for biotherapeutic applications (yogurts and other drinks are seen as supplements).


Then, we had to determine the amount of spores to include in each capsule. Spore-based probiotics typically contain 105 – 109 spores and capsules commonly contain 109 spores [4]. We expect that this amount would also allow germination and outgrowth of C. sporogenes in the gut, so we thought that each NeuroTone capsule should contain 109 spores. However, results obtained by our modelling team suggested that it might be better to use a lower amount of spores in the capsule. This would allow a smoother adaptation and prevent adverse reactions in the patient. Although a higher number of spores would result in faster establishment of the culture in the gut (and consequent faster production of DBHB), we felt that the safety and well-being of the patients should be our priority. Hence, we decided to reduce the amount of spores in each capsule to 105.


But spores are too small and cannot be administered on their own. So, what about the rest of the capsule? A previous study has shown that sodium alginate/cellulose nanofiber gel macrospheres are able to protect the probiotics from the acidity of the stomach, and then release the bacteria in the gut due to a change in pH [5]. The extra protection provided by such a capsule seemed interesting, so we decided that this would be a good option for the formulation of the capsule itself.


Finally, we researched excipients traditionally used in spore-based probiotics and selected a few options: hydroxypropyl methylcellulose, microcrystalline cellulose and magnesium stearate. These would be used as stabilising and bulking agents. Any of these options is appropriate, so our final decision would be based on results from germination assays – the component we choose should not trigger germination of our strain.


In summary, the results from our research suggest that NeuroTone should be a capsule made from sodium alginate/cellulose nanofiber gel macrospheres, containing 105 or 109 spores of the strain engineered to produce DBHB (with biosafety control mechanism) stabilised by hydroxypropyl methylcellulose, microcrystalline cellulose or magnesium stearate.

References

1. Cell Net Analyzer. 2020. url: https://www2.mpi-magdeburg.mpg.de/projects/cna/cna.html.

2. COBRApy. 2020. url: https://opencobra.github.io/cobrapy/.

3. Cañadas, I. C., et al., (2019). RiboCas: A Universal CRISPR-Based Editing Tool for Clostridium. ACS Synthethic Biology 8(6): p. 1379-1390. doi: 10.1021/acssynbio.9b00075.

4. Hong, H.A., et al., (2005). The use of bacterial spore formers as probiotics. FEMS Microbiology Reviews 29(4): 813-835. doi: 10.1016/j.femsre.2004.12.001.

5. Zhang, H., et al., (2018). A pH-responsive gel macrosphere based on sodium alginate and cellulose nanofiber for potential intestinal delivery of probiotics. ACS Sustainable Chemistry & Engineering 6(11): 13924-13931. doi: 10.1021/acssuschemeng.8b02237.