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.

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.

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.

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:

a different E. coli cloning host.
a Gram-negative replicon with a lower copy number, less of the plasmid to cause problems in the host cells.
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.

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.

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.

Team:Nottingham/Engineering

Engineering Success