iGEM 2020 — Team Nottingham

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# Team:Nottingham/Notebook

![Nottingham IGEM Logo](https://static.igem.org/mediawiki/2020/f/f0/T--Nottingham--logo_neurotone.png)

# Project Notebook

### Take a look at what we've been up to

In order to work effectively and efficiently, our team were separated into different subgroups:

![eugenia-profile](https://static.igem.org/mediawiki/2020/b/b5/T--Nottingham--eugenia_circle.png)
![saachi-photo](https://static.igem.org/mediawiki/2020/6/6e/T--Nottingham--saachi_circle.png)

**Subgroup 1: DBHB**
Hi! We’re Eugenia and Saachi and we’ve been working with our supervisors Andrew Dempster and Alex Rawson on DBHB production. Our subgroup is responsible for mapping out the synthetic DBHB production pathways in *C. sporogenes* as well as additional research on intermittent fasting and the ketogenic diet.

You can view the subgroup lab book in detail [here](https://static.igem.org/mediawiki/2020/c/c8/T--Nottingham--DBHB_subgroup_lab_book.pdf)!

![kieran-profile](https://static.igem.org/mediawiki/2020/6/6f/T--Nottingham--kieran_circle.png)
![alistair-profile](https://static.igem.org/mediawiki/2020/e/e9/T--Nottingham--alistair_circle.png)

**Subgroup 2: Routes of Administration**
Hi! we're Alistair and Kieran and as part of this subgroup, we were tasked with investigating the most suitable delivery method for NeuroTone. With supervisor Raquel Rodrigues guiding us, we tried to determine how we would deliver *C. sporogenes* to the gut microbiome. Taking advantage of its ability to form spores, many factors had to be considered including manufacturing, digestion time, germination and sporulation receptors, formulation and patient preferences.

You can view the subgroup lab book in detail [here](https://static.igem.org/mediawiki/2020/2/21/T--Nottingham--Admin_subgroup_lab_book.pdf)!

![james-profile](https://static.igem.org/mediawiki/2020/a/a1/T--Nottingham--james_circle.png)
![luke.w-profile](https://static.igem.org/mediawiki/2020/b/b5/T--Nottingham--luke.w_circle.png)

**Subgroup 3: Control**
Hi! We’re Luke W. and James, we’ve been working with our supervisor Patrick Ingle on the control mechanism for the biotherapeutic. A biological control prevents a genetically modified organism from causing harm to the environment or other organisms. They are an essential part of any GMO project that interacts with humans and control strategies need to be well thought out with multiple experiments showing their validity.

You can view the subgroup lab book in detail [here](https://static.igem.org/mediawiki/2020/8/8e/T--Nottingham--control_in_silico_lab_book.pdf)!

![aly-profile](https://static.igem.org/mediawiki/2020/6/68/T--Nottingham--aly_circle.png)
![luke-profile](https://static.igem.org/mediawiki/2020/4/4a/T--Nottingham--luke.b_circle.png)

**Subgroup 4: Modelling**
Hi! We're Luke B. and Aly and we've been tasked the mathematical modelling for the dynamic and structural models of our *C. sporogenes* biotherapeutic. Dynamic modelling handled the administration of NeuroTone, along with its interactions with the gut. Aiming to determine what time a dosage should be given, how much a dosage should be, and whether DBHB should be produced immediately, whilst the culture was still small.
With the project being entirely dry lab based, a lot of emphasis was placed on the modelling group to influence the decision about which pathway to pick. The structural model aims to answer this question by analysis which pathway is most productive, and is most likely to produce DBHB. The structural modelling took full advantage of the database the SBRC had to offer, adding metabolic formulas to ensure the reactions were balanced and feasibly possible.

You can view the subgroup log book [here](https://static.igem.org/mediawiki/2020/3/39/T--Nottingham--modelling_logbook.pdf)!

for more details about the modelling can be found on the [modelling page](https://2020.igem.org/Team%3ANottingham/Model).

### Project Progress

Week 1 (11/05/20)

1. We had our first virtual team meeting! Students and supervisors introduced themselves and discussed potential roles that
   would be best suited for them on the iGEM team. We also all prepared a project idea ready to be presented in
   the following whole team meeting.

Week 2 (18/05/20)

1. Whole team presented their project proposals.
2. Final project was decided: We plan to produce a biotherapeutic by genetically engineering a *Clostridium* strain of bacteria to increase the levels of DBHB (3-hydroxybutanoate) reaching the brain in order to
   reduce the onset of neurodegeneration.
3. Underwent iGEM safety regulation workshop with the supervisors.

Week 3 (25/05/20)

1. Further preliminary reading of natural ketone production in the body, DBHB interaction with the brain, ketoacidosis. Collated all our research to identify all aspects of our project and delegated roles and modelling objectives.
   Brainstormed ideas for outreach and integrated human practice opportunities.
2. Collated all our research to identify all aspects of our project.
3. Delegated roles and modelling objectives.
4. Brainstormed ideas for outreach and integrated human practice opportunities.

Week 4 (01/06/20)

1. Discussed carrying out a survey to find out more about the public’s perception of using GM in medicine.
2. Decided to carry out a literature review to ensure our survey would be necessary and covering new information.

Week 5 (08/06/20)

1. Discussed the different *Clostridium* strains we could use.
2. Discussed what factors would be important in selection our strain (virulence genes, genes advantageous for ketone production, preferred environment, etc.)
3. Researched the identified potential strains, breaking down the pros and cons for each one.
4. Researched the human microbiome project (a well known large source of microbe genomes) to see if there was any data we could access about the potential strains.
5. Researched Ketoacidosis.
6. Decided to start the Minecraft project to encourage community engagement in science.

Week 6 (15/06/20)

1. Shortlisted our three final strain choices.
2. Prepared presentations for each of the strain choices.
3. Considered poly-3-hydroxybutyrate and (R)-3-hydroxybutanoate as potential decisions for the ketone pathway to insert into our biotherapeutic.
4. Looked up the genes involved in the acetyl-CoA and butyryl-CoA pathways.
5. Discussed preliminary logo design options and colour schemes.
6. Brainstormed fundraising ideas for the team.

Week 7 (22/6/20)

1. We decided on *C. butyricum* as our preferred strain.
2. Presented the three potential Ketone production pathways (fatty acid metabolism, polymer pathway and butyrate pathway).
3. Split the mathematical modeling into two groups – dynamic and structural modelling.
4. Team logo finalised.
5. Discussed product logo ideas.

Week 8 (29/6/20)

1. We had genome presentations for the different strains of *Clostridium butyricum* (DKU01, KNUL09).
2. We determined the locations of the genes for the butyrate pathway in the genomes.
3. Had a presentation on acetone – risks of overdose and the process of production.
4. Researched the virulence and pathogenicity of chosen *C. butyricum* pathways.
5. Had a presentation on ethanol production, highlighting the connection between the butyrate pathway and ethanol production.
6. Discussed outreach ideas – Minecraft server and the QR code trail.
7. Had an ethics tutorial from our human practices supervisor specifically focused on running a survey and data handling.

Week 9 (06/07/20)

1. Meeting with the 2019 Nottingham iGEM team in which we discussed approached to organising and improving our project.
2. Finalised primary and secondary roles for subgroups.
3. Reached out to several companies for sponsorship.
4. Organised a meeting with Geocashing HQ to discuss collaboration over a QR code trail on campus.
5. Changed our species of clostridium to *C. sporogenes* as it was a better sporulator and already produced a neuroprotectant naturally.
6. Analysed various *C. sporogenes* strains for virulence and toxin genes to find the best candidate for our biotherapeutic.

Week 10 (13/07/20)

1. Subgroups formed (see individual subgroup notebooks for timelines of subgroup work).
2. Discussed hosting a European team meet up.
3. Had a presentation on the ethanol production from the new DBHB production pathway the team was working on.

Week 11 (20/07/20)

1. Had a discussion of the European meet up with Nemira (European iGEM Ambassador) - decided to change it to a commonwealth meet up to try and involve a more diverse range of teams.
2. Worked on branding and social media posts for a commonwealth meet up.

Week 12 (27/07/20)

1. Filmed intro videos for the team introduction
2. Discussed whether to use posters, QR codes or the Adventure Lab (from Geocashing) for our trail.
3. Discussed whether posters or videos would be best for the trail project for science education.
4. Discussed the poster presentation and the graphics and content we want to cover.
5. Discussed starting a podcast for outreach.

Week 13 (03/08/20)

1. Shot the videos for Nemira’s UK video for the global meetup.
2. Discussed entering the Tough Mudder challenge to raise money and awareness for a neurodegenerative disease charity.
3. Meeting with Subin Saji (experienced podcaster and friend of the team) about how to run and edit a successful podcast.

Week 14 (10/08/20)

1. Made the podcast logo.
2. Started recording vlogs.
3. Finished the MolecularCloud livestream presentation.
4. Finalised the platforms we would upload the podcast on.
5. Each team member wrote a personal paragraph for the meet the team wiki page.

Week 15 (17/08/20)

1. Recorded first podcast (with our Nottingham student team).
2. Contacted the Paris team - had a meeting to discuss opportunities for collaboration.
3. Made a general poster with the basic guidelines to recording a podcast to send out to teams. Check out the poster [here!](https://static.igem.org/mediawiki/2020/d/d7/T--Nottingham--podcast_guide_poster.pdf)
4. Made a podcast guide on how to start up your first podcast!
5. Minecraft promotional video produced.
6. Finalised product name – NeuroTone.

Week 16 (24/08/20)

1. Discussed our approach to the Science Education and Modelling Special awards.
2. Discussed ways to encourage science communication by setting up Reddit forums and accepting questions about Synthetic Biology from social media to be answered on out podcast recordings.
3. Meeting with Kings College London iGEM team to discuss potentially fulfilling partnership criteria together.
Recorded a podcast episode with the Paris-Bettencourt Team.

Week 17 (31/08/20)

1. Posted an advert for the Minecraft video on our social media. Here's the [Minecraft video](https://www.youtube.com/watch?v=wRHX9zJGMEE) on our youtube channel!
2. Finalised the project promotion video.
3. Finalised the logo and our team hoodie designs.
4. Recorded a podcast with Kings College London team.

Week 18 (07/09/20)

1. Recorded the strain choice video clips.
2. Recorded a podcast with Warwick University team.

Week 19 (14/09/20)

1. Strain choice video finalised.
2. Decided to post an ad for the podcast series on our social media.
3. Recorded a podcast with Denmark team.

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iGEM Nottingham *2020*

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Nottingham/Engineering

![Nottingham IGEM Logo](https://static.igem.org/mediawiki/2020/f/f0/T--Nottingham--logo_neurotone.png)

# Engineering Success

The experiments for our project have be designed using the fundamental principles of synthetic biology and the engineering design cycle:
**research, design, build, test, learn, improve** and **research**  of our sporulation methods, synthetic pathways and control mechanisms.

![design-cycle](https://static.igem.org/mediawiki/2020/5/51/T--Nottingham--engineering_cycle.png)

Experiments to build and test have been planned, unfortunately with the lab being closed to us, we can only plan for **potential unexpected results**!
Our supervisors have been telling us about the (many) potential unexpected results we would expect with a lab based project, here we will detail our workflow and the most common problems we would likely face and the **potential solutions**!

A fundamental first principle for our project, is production and administration of our biotherapeutic. The **Administration subgroup** have **researched** to plan experiments to **design** and **test** the most effective ways to initiate sporulation in *C. sporogenes*, which will be used to administer NeuroTone, ensuring delivery to the gut. They would also have been harvesting spores and conducting germination experiments.

At the very core of our project, we need our biotherapeutic to produce the therapeutic molecule DBHB. The **DBHB subgroup** have **researched** to plan experiments to **design** and **build** plasmids and integration steps for DBHB production in our strain of *Clostridium sporogenes* and then **test** DBHB production.

Integral to the project, any biotherapeutic needs to have appropriate control mechanisms to ensure safety to the patient and containment of the organism. The **Control subgroup** have planned experiments to **design** and **build** plasmids to integrate control mechanisms in to the chromosome our strain of *C. sporogenes* and then **test** these mechanisms.

Optimising Sporulation Conditions and Yields in *C. sporogenes* NCIMB 10969

The **Administration subgroup** have  **designed** experiments to test different media and culture conditions to produce the maximum yield of spores. Cultures will be set up in different media conditions and sporulation assays carried out.

**Potential Unexpected Result** –sporulation levels are low for  **test**  conditions.

**Potential Solution** –  **Redesign** of the experiment to optimise media for sporulation and repeat sporulation assays to  **test** our culture conditions again.

Once the optimal conditions for the best yield of spores is identified, The  **Administration subgroup** will then proceed to culture and harvest spores. These spores will then be enumerated and be subjected to germination assays to ensure viability. Germination assays will also be carried out with components of the capsule used to deliver the spores to the gut, to ensure that none of the components of the capsule will trigger germination early.

**Potential Unexpected Result** – Capsule component induces germination of the spores.

**Potential Solution** –  **Learning** from this outcome, we would re-evaluate which capsule to use and repeat our germination  **test**.

Construction of Vectors

For **DBHB** production, two pathways have been **designed** informed by data produced by our very own modelling team.

The plasmids we will use to test our synthetic pathway will be **built** using the homology based NEB HiFi assembly method. Each gene, including regulatory elements will be amplified with PCR, including complementary sequence overhangs. The vector backbone will be linearised and overhangs added, also using PCR. These fragments will then be assembled in to the pMTL82151 shuttle plasmid using the NEB HiFi reaction mixture, which includes bothexonuclease and ligation activity.

The resultant reaction mixtures will then be transformed in to an *Escherichia coli* cloning host and selected for by the desired antibiotic marker. Potential plasmids will be confirmed using Sanger sequencing.

For the **Control** aspect of our project, the plasmids we will use to create the conditional sporulating strain will also be **built** using the homology based NEB HiFi assembly method. The sequence for the tet inducible promoter will be amplified with PCR, including complementary sequence overhangs, along with homology arms complementary to enable insertion up stream of each sporulation gene. The inducible regulatory sequence will be assembled between these homology arms in to the vector backbone will be linearised and overhangs added, also using PCR.

**Potential Unexpected Result** – The HiFi assembly method may not result in the construction of a viable plasmid, which may be observed after transformation of the *E. coli* host, i.e. no colonies observed on selective media.

**Potential Solution** – repeat the experiment, **learn** and **redesign** the HiFi primers if necessary. It may also be that some of the genes we are assembling could be toxic to the host, resulting in a failed assembly. This could potentially be solved:

1. a different *E. coli* cloning host.
2. a Gram-negative replicon with a lower copy number, less of the plasmid to cause problems in the host cells.
3. incubation of *E. coli* transformants at 30 °C instead of 37 °C, to reduce accumulation of these potential toxic gene products.

**Potential Unexpected Result** – Transformant colonies are observed and plasmids isolated, but the sequences may have mutations.

**Potential Solution** – This could also be due to the potential toxicity of the genes we are introducing and so the solutions listed above could also apply. **Learning** from this, another solution would also be to screen a larger number of clones.

Conjugal Transfer of Constructed Plasmids

Each of the assembled plasmids will then be transferred to our *Clostridium sporogenes* strain via an *E. coli* conjugal donor. Conjugation mixtures will be plated on to media containing antibiotics to select against the *E. coli* donor and to select for the plasmid.

**Potential Unexpected Result** – No transconjugant colonies observed.

**Potential Solution** – Optimisation of conjugation protocol, by increasing mating time.

**Potential Unexpected result** – Background E. coli colonies observed on selection plates.

**Potential Solution** – Remake the working antibiotic stocks and make sure the media is made with correct volume of antibiotics added.

Once we have confirmed the presence of our plasmids in our strain, the **DBHB subgroup** will proceed to culture and **testing** for DBHB production by gas chromatography.

**Potential Unexpected Result** – Not enough or no DBHB is produced!

**Potential Solution** – This is why we are testing a number of different promoter variants. If these prove to not work, we will return to the drawing board and **redesign** our operons to use different upstream regulatory genes and RBS sequences. We will then repeat the processes of **building** and **testing,**  hopefully without returning to the **redesign** step again!

The **Control subgroup** will proceed to integrating the tet inducible promoter in to the chromosome upstream of the identified sporulation genes.

Integration in to the Chromosome of *C. sporogenes* NCIMB 10696

The **DBHB subgroup** will then perform experiments to integrate the most promising pathway in to the chromosome of our strain of *C. sporogenes* using the SBRC’s CRISPR pRECas system. These integration plasmids will be **built** using NEB HiFi in the process detailed above, with areas of sequence homology to where we want to integrate our operon – called homology arms.

The **Control subgroup** will perform similar integration experiments to integrate the regulatory tet inducible promoter upstream of the sporulation genes as detailed previously.

As with the plasmids, we will screen for the presence of these genes after transferring in *C. sporogenes* and inducing pRECas CRISPR mediated integration, but this time we are screening for integration of our pathway genes in to the chromosome using primers which flank the integration locus and primers which bind to the genes to be integrated.

The **Control subgroup** will make four individual sporulation mutants and a fifth mutant, incorporating all of these integrations in to a strain with all four genes under the control of the tet inducible promoter.

**Potential Unexpected Result** – The desired genes and regulatory sequences appear to be unable to integrate in to the chromosome.

**Potential Solution** – Firstly, we would repeat the integration experiment and if this still fails, the next solution to try would be to use a second synthetic guide RNA (sgRNA), if the first is inefficient at directing the Cas9 to select for integrants. Another potential solution would be to **redesign** the homology arms.

Once integration is confirmed, we will aim to achieve plasmid loss before **testing** our integrants.

The **DBHB subgroup** will again test for DBHB production with gas chromatography and the **Control subgroup** will perform sporulation assays, assisted by the **Administraton subgroup**, with and without inducer anhydrotetracycline.

**Potential Unexpected Result** – sporulation proceeds in the **Control** strain.

**Potential Solution** – This is extremely unlikely, but this is why we are choosing four sporulation targets, with the master regulator spo0A a proven target to completely abolish sporulation. The strain with multiple integrations engineered should provide a synergistic effect in the ablation of sporulation.

**Potential Unexpected Result** – Not enough DBHB is produced.

**Potential Solution** – At this stage this would be highly unlikely, but could be due to the locus where we are integrating the DBHB production operon. The pyrE locus was chosen as it is a tried and tested region of the genome for this purpose, as demonstrated in the SBRC and by the 2019 Nottingham iGEM team. A potential solution would be to **redesign** our homology arms in the integration vectors to integrate at a different genome locus.

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iGEM Nottingham *2020*

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# Team:Nottingham/Experiments

![Nottingham IGEM Logo](https://static.igem.org/mediawiki/2020/f/f0/T--Nottingham--logo_neurotone.png)

# Experiments

Since our labs were closed during our project, we have come up with all the potential wet lab experiments we could have done if the circumstances were different.

### DBHB and Control subgroup protocols:

**Protocol 1** - Genomic DNA extraction using GenElute™ Bacterial Genomic DNA Kit by Sigma Aldrich

1. Pellet 1 mL of a liquid overnight culture of the bacterial strain by centrifugation at 13,000 rpm and resuspend in 200 µL lysozyme (10 mg/ml) and incubate for 30 minutes at 37 °C.

2. Lyse the cells by the addition of 20 µL Proteinase K (20 mg/ml) and 200 µl lysis solution C and incubate for 10 minutes at 55 °C.

3. Prepare the column by adding 500 µL of the Column Preparation Solution to each pre-assembled GenElute Miniprep Binding Column in a 2 mL collection tube. Pass through by centrifugation at 13,000 rpm for 1 minute and discard eluate.

4. Add 200 µL of ethanol (95–100%) to the incubated cell lysate (from step 3), mix thoroughly and load into the prepared binding column. Centrifuge a 13,000 rpm for 1 minute and discard eluate in collection tube.

5. Wash the loaded column by adding 500 µL of Wash Solution 1, centrifuge for 1 minute at 13,000rpm. Wash for a second time by adding 500 µL of Wash Solution 2 to the column and centrifuge for 3 minutes at 13,000 rpm ensure the column is dry. Discard eluate in the collection tube after each wash step.

6. Elute the DNA by pipetting 200 µL of the Elution Solution directly onto the centre of the column, incubate for 5 minutes at room temperature and centrifuge for 1 minute at 13,000 rpm.

**Protocol 2** – PCR for amplification of DNA fragments using High fidelity KOD Hot Start DNA polymerase

Table 1 - Standard PCR Reaction Setup using KOD Hot Start Master Mix. Starting template amount varies depending on template type used (B); Plasmid DNA - 10 ng, Genomic DNA – 100ng. Volume of dH20 (A) added to make reaction up to 20 µL.

| Component | Volume (total 20 µL reaction) |
| --- | --- |
| KOD Hot Smart DNA Polymerase Master Mix (0.04 U/ µL) | 10 µL |
| Primer 1 (µM) | 0.6 µL |
| Primer 2 (µM) | 0.6 µL |
| Template DNA | B |
| Deionized H2O | A |

Table 2 - Thermocycling Conditions. TA (annealing temperature) should be lowest primer Tm (melting temperature)

| Step | Target Size | | | |
| --- | --- | --- | --- | --- |
| <500bp | 5000-1000bp | 1000-3000bp | >3000bp |
| 1. Polymerase activation | 95 °C - 2 mins | 95 °C - 2 mins | 95 °C - 2 mins | 95 °C - 2 mins |
| 2. Denaturation | 95 °C – 20 s | 95 °C – 20 s | 95 °C – 20 s | 95 °C – 20 s |
| 3. Annealing | TA °C – 10 s | | | |
| 4. Extension | 70 °C – 20 s/kb | 70 °C – 20 s/kb | 70 °C – 20 s/kb | 70 °C – 20 s/kb |
| Repeat steps 2. - 4. | 40 cycles | | | |

**Protocol 3** – Gel electrophoresis to separate DNA fragments by length

1. Make 1% agarose (1 g agarose in 100 ml TAE buffer and heated in a microwave until clear and left in a 50 °C water bath for 30 mins to cool).

2. Add SYBR Safe (DNA stain) to the molten agarose (5 µl/100ml of agarose) and pour into a cast, inserting an appropriately sized comb.

3. Let set for around 20 minutes.

4. Remove the comb and gel in cast from its holder and place into a gel running tank with TAE buffer ensuing the entire gel in submerged.

5. Load DNA samples by pipette into the gel wells (including a DNA ladder in the first well (e.g. NEB 1 kb plus ladder).

6. Place the lid on the gel tank, attach wires to correct terminals in power pack and run at 100 Volts and 40 minutes.

7. Image gel with an imaging machine and if required cut bands using a clean scalpel for gel for purification (see protocol 4).

**Protocol 4** - Gel extraction using a QIAGEN QIAquick Gel Extraction Kit

All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge.

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.

2. Weigh the gel slice and add 3 volumes Buffer QG to 1 volume 1% agarose gel (100 mg gel ~100 µl).

3. Incubate at 50 °C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 µl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.

4. Add 1 gel volume isopropanol to the sample and mix.

5. Place a QIAquick spin column in a provided 2 ml collection. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min.

6. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes >800 µl, load and spin/apply vacuum again.

7. To wash, add 750 µl Buffer PE to QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube.

8. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.

9. To elute DNA, add 30 µl water to the center of the QIAquick membrane, let the column stand for 1 min and centrifuge the column for 1 min.

**Protocol 5** - Quantification of DNA using Thermo Scientific NanoDrop – a spectrophotometer.

1. Clean the upper and lower optical surfaces of the microvolume spectrophotometer with clean deionized water and a clean, dry, lint-free lab wipe.

2. Open the NanoDrop software and select the double stranded nucleic acid application.

3. Perform a blank measurement by dispensing 1 µL of clean deionized water (same as used to eluted kit column) onto the lower optical surface. Lower the lever arm and select "Blank" in the Nucleic Acid application.

4. Once the blank measurement is complete, clean both optical surfaces with a clean, dry, lint-free lab wipe.

5. Dispense 1 µL of nucleic acid sample onto the lower optical pedestal and close the lever arm. Select "Measure" and the software will automatically calculate the nucleic acid concentration and purity ratios which is displayed on equipment’s screen.

**Protocol 6** - NEBuilder HiFi DNA Assembly Reaction Protocol for fast and efficient “scarless” assembly of multiple gene fragments.

Table 3 – Standard HiFi DNA assembly reaction setup

|  | 2-3 Fragment assembly | 4-6 Fragments assembly |
| --- | --- | --- |
| DNA Molar Ratio | vector : insert = 1:2 | vector : insert = 1:1 |
| Total amount of Fragments | X µL (0.03-0.2 pmols) | X µL (0.2-05 pmols) |
| NEBuilder HiFi DNA Assembly Master Mix | 10 µL | 10 µL |
| Deionized H20 | 10- X µL | 10- X µL |
| Total reaction volume | 20 µL | 20 µL\* |

1. Setup the reaction above on ice.
(pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons))
\*- If greater numbers of fragments are assembled, increase the volume of the reaction, and use additional NEBuilder HiFi DNA Assembly Master Mix.

2. Incubate samples in a thermocycler at 50 °C for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled). Following incubation, store samples on ice or at –20 °C for subsequent transformation.

**Protocol 7** - Transformation into NEB chemically competent E. coli cells

1. Thaw chemically competent cells on ice and transfer 50 µL of competent cells to a 1.5 ml microcentrifuge tube (if necessary).

2. Add 2 µL of assembled product to NEB competent cells and mix gently by pipetting up and down or flicking the tube 4–5 times. Place the mixture on ice for 30 minutes. Do not mix.

3. Heat shock at 42 °C for 30 seconds. Do not mix.

4. Transfer tubes on ice for 2 minutes.

5. Add 950 µL of room temperature SOC media to the tubes and incubate at 37 °C for 60 minutes, shake vigorously (250 rpm).

6. Warm selection plates (with appropriate antibiotics) to 37 °C and spread 100 µL of the cells onto the plates with.

7. Incubate plates at 37 °C until colonies appear (usually after 24 hours).

**Protocol 8** – Preparation of agar plates containing appropriate antibiotic(s) and/or supplements

1. Label approximately 20 plates per 400 mL bottle of agar media.

2. Melt a 400 mL Duran bottle of agar media in a microwave on low power (for 10-15 minutes) with the cap unscrewed.

3. Leave the bottle in a 50 °C water bath for around 30 mins to allow the media to cool.

4. Under a bunsen burner (to keep aspectic) add required volume of antibiotic(s)/supplements and pour media into labelled plates until the surface is covered.

5. Let plates solidify on the bench and before use dry plates at 37 °C for ~40 minutes. Store any extra plates at 4 °C upside down to ensure condensation doesn’t collect on the agar surface.

**Protocol 9** - Colony PCR to screen for successful transformants or genomic mutants using DreamTaq™ Green PCR Master Mix.

1. Pick a single colony using a small sterile 1 µL inoculation loop and resuspend in 10 µL of dH2O.

2. Use 1 µL of this colony suspension in the reaction setup outlined below (table 4) and transfer tubes to thermocycler following the steps in table 5 – this includes a 10 minute denaturation step to lyse the cells.

Table 4 -Standard PCR Reaction Setup using Dreamtaq™ Green PCR Master Mix for colony PCR.

| Component | Volume (total 20 µL reaction) |
| --- | --- |
| DreamTaq™ Green PCR Master Mix (2X) | 10 µL |
| Primer 1 (10 µM) | 1 µL |
| Primer 2 (10 µM) | 1 µL |
| Template DNA | 1 µL colony suspension |
| Deionized H2O | 7 µL |

Table 5 -Thermocycling Conditions for colony PCR. TA (annealing temperature) should be lowest primer Tm (melting temperature)

| Step | Temperature | Time | Number of cycles |
| --- | --- | --- | --- |
| Initial denaturation | 95 | 10 mins | 1 |
| Denaturation | 95 | 30 s | 40 |
| Annealing | TA | 30 s |
| Extension | 72 | 1 min/kb |
| Final extension | 72 | 10 mins | 1 |

**Protocol 10** - Plasmid extraction (Miniprep) using a QIAGEN Miniprep kit.

1. Pellet 1-2 mL of an overnight bacterial liquid culture by centrifugation at maximum speed (~13,000 rpm) and remove the supernatant.

2. Resuspend pellet in 250 µL P1 (Resuspension Buffer) and vortex.

3. Lyse cells by adding 250 µL of P2 (Lysis Buffer) and mix by inverting the tube 4-6 times. Do not vortex.

4. Neutralize the lysate by adding 350 µL of N3 (Neutralization Buffer) and mix thoroughly by inverting the tube 4–6 times.

5. Centrifuge for 10 minutes at maximum speed.

6. Transfer the supernatant from step 4 to a QIAprep 2.0 spin column, centrifuge for 1 minute and discard flow-through.

7. Wash the column by adding 500 µL Buffer PB (wash Buffer 1), centrifuge for 1 min, discard flow-through and repeat this step with 750 µL of Buffer PE (wash buffer 2). Discard all flow-through and centrifuge at max speed for an additional 1 minute to remove residual wash buffer.

8. Place spin column in a clean 1.5 mL microcentrifuge tube. To elute plasmid DNA add 50 µL of dH2O to the centre of the column leave to stand 1 minute and centrifuge for 1 minute.

**Protocol 11** - Transformation of plasmid DNA by electroporation, using a Bio-Rad MicroPulser ™ Electroporator

1. Thaw the electrocompetent *E. coli* cells to be used on ice – in 50 µL aliquotes.

2. Add ~200-500 ng of plasmid DNA into the tube with the competent cells and transfer the mixture to a 0.2 cm Bio-Rad electroporation cuvette.

3. Transfer the cuvette on ice to the electroporator and set to the appropriate setting.

4. Load the cuvette into the electroporator, pulse (with a read out >5.0), retrieve cuvette and immediately add 400 µL of SOC media (Invitrogen™). Transfer the whole mixture to a labelled microcentrifuge tube and incubate at 30 °C, shaking at 250 rpm, for at least 1 hour to recover.

5. Plate a suitable volume (200 µL) onto the appropriate agar selection plates and incubate at 30 °C until colonies appear (usually after 24 hours).

**Protocol 12** - Conjugation into *C. sporogenes*

1. Inoculate vectors transformed into donor *E. coli* CA434 in 5 ml of LB (+2.5 µL Chloramphenicol) at 30 °C shaking at 250 rpm overnight. In the anaerobic cabinet also inoculate 1 mL of reduced TYG with *C. sporogenes* and leave to incubate overnight.

2. In the morning pellet the *E. coli* by centrifuging 1 mL of this overnight culture at 4000 rpm for 1 minute.

3. Wash pellet by resuspending in 400 µL of PBS and centrifgue at 4000 rpm for 1 minute. Discard supernatant and repeat one more wash.

4. Transfer the washed *E. coli* donor to the anaerobic cabinet.

5. Gently resuspend the *E. coli* pellet in 200 µL of the *C.  sporogenes* overnight culture and plate the mixture onto a TYG plate supplemented with glucose (0.5% v/v final concentration) in 5 or 6 dots. Leave the bacteria to mate for at least 7 hours or overnight.

6. After the mating time harvest all the cells with a sterile 10 µL loop and gently resuspend in 500 µL of reduced PBS in a microcentrifuge tube.

7. Plate 100 µL this PBS mixture onto pre-reduced TYG -Thiamphenicol – D-Cylcoserine selective plates (with final concentrations of thiamphenicol - 15 µg/mL, D-Cylcoserine - 250 µg/ml) and leave to incubate in the anaerobic cabinet.

8. After 48 hours trans-conjugant colonies should appear.

**Protocol 13** - Plasmid loss of confirmed mutants

1. Inoculate 10 mL of reduced TYG broth only in the anaerobic cabinet with a confirmed transconjugant mutant.

2. Leave the culture for 48 hours to give the bacteria time to lose the plasmid.

3. Serially dilute the culture down to 10-4 in reduced PBS and spread on a reduced TYG only agar plate to obtain single colonies.

4. Patch plate these single colonies onto first TYG only plate and then TYG plate supplemented with Thiamphenicol (15 µg/mL) and leave to incubate overnight.

5. Colonies that have lost the plasmid will only be able to grow on the TYG only agar plates.

**Protocol 14** - Primers Design using SnapGene

The following step were followed when designing our primers

1. Each primer was at least 18bp (and less then 100 bp) long and an annealing temperature (TA) ensured to be between 55 °C and 72 °C.

2. A G-C clamp was desirable when looking for primer sequences - the last base of the primer (at the 3’ end) is a G/C base pair.

3. The primer melting temperature (Tm) was checked online by a Tm calculator- this shows the temperature that duplex will melt at. The TA is typically 5 °C lower than Tm calculator value.

4. Secondary structures were checked online at the mfold web server- ensuring the delta G values are positive (spontaneously secondary structures will not happen at the desired temperature). If secondary structures predicted made sure the Tm value of the secondary structures is significantly lower than your TA.

5. To amplify DNA primers were designed in pairs, with the forward and reverse primers annealing to the opposite DNA strand.

6. The NEB assembly Tool was used to design HiFi primers. This programme added complementary overhangs to the 5’end of a primer to enable HiFi assembly.

**Protocol 15** - Measuring the growth Optical Density (OD) of bacterial cultures to determine the bacterial population density (monitor the growth of the culture). As *C. sporogenes* is an anaerobic organism all growth must occur within an anaerobic cabinet.

1. Inoculate reduced (left in an anaerobic cabinet for at least 4 hours to allow for all the oxygen to be lost) liquid media with the bacterial strain from a frozen stock. Adding appropriate antibiotic at the required concentration if needed. Leave to growth overnight.

2. Dilute the overnight culture 1:100 into fresh liquid media – this is t = 0 hours.

3. Pipette a 200 µL sample of this culture into well of a 96 well plate (in triplicate). Put the 96 well plate into a microplate absorbance reader and measure at OD600.

4. Incubate the culture for usually 72 hours, taking samples (as described for t=0) every 2 hours from t=0 to t=12 and then at t=24 hours, t=48 and t=72 hours. When the OD of the culture reaches >0.8, dilute the samples by a factor of 10 (20 µL of sample, 180 µL TYG) in the plate well.

5. These recorded OD values can then be plotted as a ‘growth’ curve, with time on the x-axis and OD on the y-axis.

**Protocol 16** - Gas Chromatography (GC) to analyse the levels DBHB in the supernatant at different timepoints – preform in tandem with culture OD measurements.

1. Aliquot a 1 mL of each sample culture into a respectively labelled 1.5 mL microcentrifuge tube and centrifuge for 1 minute at 16,000 rpm.

2. Pipette the supernatant into fresh labelled 1.5 mL microcentrifuge tubes, taking care not to disturb the pellet which has formed.

3. Store the resultant supernatant samples -80 °C until all timepoint samples have been collected.

4. Take samples every 2 hours from inoculation of culture (t=0) to t = 12 and then at t=24 hours and t=48 hours.

5. Once all samples have been taken, thaw all samples and follow the standard GC protocol which is used at your institute.

### Routes of Administration subgroup protocols:

**Protocol 1** - **Lab scale** production and purification of *Clostridium sporogenes* spores using a sucrose gradient

1. Set up a culture of *C. sporogenes* (TYG medium, grow at 37 °C in the anaerobic cabinet).

2. Leave culture in the cabinet for at least 5 days to induce sporulation by starvation.

3. Heat-treat culture (80 °C, 20 min) to kill any remaining vegetative cell (spores survive the heat treatment). After heat-treatment, allow culture to cool down.

4. Divide culture into several tubes and spin. Discard supernatant.

5. Wash each pellet with water and spin. Discard supernatant.

6. Wash each pellet with ethanol and spin (ethanol should kill remaining vegetative cells but not spores). Discard supernatant.

7. Wash again with water to remove ethanol residues. Spin and discard supernatant.

8. Resuspend final pellet in water.

9. Pour sucrose solution (50% w/v) into Falcon tubes.

10. Gently pipette spore suspension on top of the sucrose.

11. Gently place tubes in centrifuge and spin. The vegetative cells and debris will be collected at the interface and in the gradient, while the spores will move through the gradient and form a pellet at the bottom of the tube.

12. Gently remove the cell debris from the interface and then the rest of the solution, leaving the spore pellet.

13. Wash spore pellets with water. Spin and discard supernatant.

14. Resuspend spores in water.

**Protocol 2** - **Industrial scale** protocol for how we would produce and sterilise our spores, using *Bacillus coagulans* as an example.

1. Take pure culture of *Bacillus coagulant*.

2. Incubate to sterilise seed medium.

3. Confirm the purity through microscopy.

4. Add water, supernatant of Corn Steep Powder, MnSO4, CaCO3, Dextrose and Ammonium Sulphate.

5. Adjust pH with Ortho-Phosphoric Acid.

6. Sterilise for 20-25 mins at 1.5 ± 0.2 bars.

7. Cool the media to 37 °C - 29 °C.

8. Inoculate with the seed inoculum.

9. Agitate and provide sterile aeration to the fermentation medium.

10. Incubate at 37 °C - 39 °C for 35-37 hrs.

11. Centrifuge at 13,000 - 15,000 rpm.

12. Add sterile water in 1:1 ratio.

13. Filter the slurry through SS sterile mesh.

14. Spray dry the filtered slurry at 115 °C inlet temperature and 55 °C outlet temperature.

15. Dialate using Maltodextrin

**Protocol 3** - Potential industrial protocol for *C. sporogenes*

1. Inoculate sterile, reduced seed medium with *C. sporogenes*.

2. Incubate for 24 hours under anaerobic conditions, at 37 °C. Confirm purity of the culture through microscopy.

3. Prepare appropriate medium (previously selected by performing sporulation assays and evaluating spore formation), sterilise it and reduce it.

4. Inoculate medium with pure *C. sporogenes* from the seed culture.

5. Incubate under anaerobic conditions, at 37 °C, for 5 days (or the optimal time previously determined by performing sporulation assays).

6. Harvest cells by centrifugation.

7. Add sterile water and filter the slurry through SS sterile mesh (previous assays would have to be performed to determine if the mesh would also allow the purification of *C. sporogenes* spores).

8. Spray dry the filtered slurry and use the concentrated spay dried powder in the formation of the pill.

**Protocol 4** - **Lab scale** protocol for sporulation assay

Note: sub-culturing ensures that there are no spores at the beginning of the assay (time point 0h), only vegetative cells.

1. Set up *C. sporogenes* overnight culture (TYG broth).

2. Sub-culture into fresh TYG (1:100) and incubate for 3h.

3. Sub-culture again into fresh TYG (1:100).

4. Immediately take 0h sample and divide in 2 aliquots.

5. Heat-treat one of the aliquots (80 °C, 20 min).

6. Serially dilute both aliquots (heat treated, and non-heat treated).

7. Plate appropriate dilutions onto TYG plates.

8. Repeat steps 4 to 7 for each time point (24 h, 48 h, 72 h, 96 h and 120 h).

9. For each time point, count colonies after approx. 24 h of growth.

10. Calculate CFU/mL:

CFU/mL = N x D x 1000 µL / V(µL)

1. N: number of colonies
2. D: dilution factor
3. V: volume plated

**Protocol 5** - Germination assay

1. Set up *C. sporogenes* overnight culture (TYG broth).

2. Sub-culture into fresh TYG (1:100) and incubate for 120 h to allow spore formation.

3. Heat-treat culture (80 °C, 20 min) to kill vegetative cells.

4. Purify the spores (follow spore production and purification protocol, using sucrose gradient).

5. At time point 0 h, resuspend the spores in the germinant being tested to an OD600 of 1.0.

6. Monitor OD600 at several time points: 0 h, 2 min, 5 min, 10 min, 15 min, 30 min, 45 min, 60 min and 90 min. This can be done in the CLARIOstar plate reader.

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iGEM Nottingham *2020*

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Nottingham/Results

![Nottingham IGEM Logo](https://static.igem.org/mediawiki/2020/f/f0/T--Nottingham--logo_neurotone.png)

# 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]](#section2), and COBRApy [[2]](#section2) to determine which pathway was superior. The two pathways are discussed in more detail on the [Model](https://2020.igem.org/Team%3ANottingham/Model) and [Design](https://2020.igem.org/Team%3ANottingham/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](https://2020.igem.org/Team%3ANottingham/Human_Practices)) 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](https://2020.igem.org/Team%3ANottingham/Design).

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.

![](https://static.igem.org/mediawiki/2020/4/48/T--Nottingham--DBHB_Results_Figure1.jpg)

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]](#section2) (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.

![](https://static.igem.org/mediawiki/2020/2/28/T--Nottingham--Results_DBHB2.jpg)

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](https://static.igem.org/mediawiki/2020/3/3b/T--Nottingham--DBHB_lab_book.pdf)) - 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.

![](https://static.igem.org/mediawiki/2020/3/32/T--Nottingham--Results_DBHB3.jpg)

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]](#section2). 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]](#section2). 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](https://doi.org/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](https://doi.org/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](https://doi.org/10.1021/acssuschemeng.8b02237).

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iGEM Nottingham *2020*