Registration is now open. The deadline for submitting abstracts (and for early registration) is Wednesday 6 September 2023.
Synthetic biology is a maturing field at the confluence of the biosciences, physical sciences, information technology, and engineering and is rapidly beginning to demonstrate valuable solutions to some of our most pressing global challenges in healthcare, agriculture, sustainability and the environment.
SBUK 2023 will bring the synthetic and engineering biology communities together in Bristol, a city with a rich and long-standing history in engineering and a vibrant research community in synthetic biology, to share recent scientific advancements across the field. Conference themes will include biomolecular design and engineering; cell and system-level design; synthetic and minimal cells; data-centric bioengineering, and applications across industry.
The diverse and inclusive programme will include opportunities for networking, and an emphasis will be placed on ensuring the event offers early career researchers with the vital opportunities needed to build their wider research networks with leading experts in the field, as well as gain insights into careers in industry, SMEs, policy, academia and innovation delivery.
The School of Biochemistry was delighted to host Thangam Debbonaire, Labour MP for Bristol West and Shadow Leader of the House of Commons, on 9 June 2023.
Thangam toured the labs of Mark Dodding and BBI Director Dek Woolfson, where group members demonstrated their experiments to visualise motor protein activity in vitro and inside cells.
Jessica and Thangam were joined by VC Evelyn Welch and researchers Laura O’Regan, Kate Kurgan, Bernadette Carroll, Rachel Curnock, Kirsty McMillan and Emma Jones for lunch. It was a welcome opportunity to celebrate the contributions of women in science and to address barriers for women following careers in STEM.
Congratulations to Dr Jessica Cross, an ESPRC Doctoral Prize Fellow working in the Dodding/Woolfson labs, for making the shortlist of the L’Oreal-UNESCO For Women in Science Rising Talent Program, and being one of two highly commended applicants in the Physical Sciences category. Jessica visited 10 Downing Street, where she met George Freeman MP (Minister of State for Science, Research & Innovation), and has attended a training day for shortlisted candidates at the Royal Society, and a reception at the House of Commons.
Among the 170 attendees at the reception were MPs, academics and representatives from L’Oréal and UNESCO. These events have given Jessica and the others the chance to showcase their research, raise awareness of the important contribution of women in science, and discuss barriers for women in STEM and the need for policy change.
Jessica said: “I am pleased and honoured to be recognised as a highly commended candidate by the L’Oreal UNESCO For Women in Science Program. It has been a fantastic opportunity to share our research and to network with inspiring women in science. This program is a good example of showcasing female talent in science and offering role models to the next generation of science leaders.”
As part of the ADDovenom team, the post-holder will utilize state-of-the-art selection/evolution technology (Ribosome Display) to generate high-affinity binders (nanobodies and new scaffold proteins) that neutralise snake venom toxins. This project to develop new, safe and efficient antivenom to treat snakebites is an international collaboration with Liverpool School of Tropical Medicine, University of Liege, University of Aix-Marseille, and iBET.
Experience with protein expression and purification is essential. Experience with biochemical and biophysical analysis of proteins and RNA isolation and preparation, and/or current molecular biology methods is a plus.
Research activity in the Woolfson group spans rational and computational peptide and protein design; the production of peptides and proteins using both synthetic and recombinant approaches; the biophysical and structural characterization of these molecules; and the applications of the resulting de novo peptide/protein modules to address problems and challenges in cell biology and biotechnology.
The postholder is expected to become actively engaged in these research activities, and would be encouraged to lead one of the group’s research programmes.
The primary responsibilities of this post are to ensure the smooth running of experimental aspects of the Woolfson research lab; to take responsibility for the day-to-day running of research in the Woolfson lab; and to help oversee the training of undergraduate and new post-graduate students, which number up to 10 each year. There will be considerable opportunity for the postholder to develop new research projects in peptide/protein design and synthetic biology with Professor Woolfson, including co-writing grant proposals and co-supervising research grants in the lab. This could extend to helping forge new collaborative research links and endeavours both across the University and with international collaborators.
Further, together with Professor Woolfson, the post-holder would be expected to contribute to undergraduate teaching in the School of Chemistry, predominantly on its first-year and Life Chemistry modules, and there will be some associated administrative duties.
There will be much more to come throughout 2023, and we are looking forward to beginning the year with the BrisEngBio Annual meeting, and BrisEngBio Connect Partnership and Networking Event.
Congratulations to the team at Halo Therapeutics, who have won the ‘BioSeed ‘One to Watch’ Award for Therapeutics’ at the OBN Awards. Halo is developing pan coronavirus antivirals for home use by COVID-19 patients, based on groundbreaking discoveries made during the lockdowns. The award was collected by Prof Imre Berger, who is co-founder and CSO of Halo, and a Co-Director of the Bristol BioDesign Institute. Halo was founded on IP generated through BrisSynBio.
Prof Imre Berger collecting the OBN Award on behalf of Halo Therapeutics (Photo: OBN UK)
This technology relies on the ability of the CRISPR-Cas13a protein to detect specific sequences of RNA when coupled with a crRNA guide. crRNA guides can be designed to detect any unique target sequence. When the protein-RNA complex finds the target sequence, it becomes activated and begins to cleave any RNA molecules in its path, including RNA ‘reporter’ probes which ‘light up’ when cleaved.
The SHERLOCK system uses this approach to indicate the presence or absence of specific RNA sequences in samples. Please see the original publications for a schematic explaining this system in more detail [Kellner et al., (2019); Gootenberg et al., (2017)].
Producing Cas13a, a protein that can detect specific sequences of RNA
We began by transforming E. coli with the required plasmid (Addgene #90097) and then grew 8 litres of bacterial culture expressing the protein overnight (see below), before lysing the cells to release the protein. The Cas13a was then purified by affinity, ion-exchange, and finally size-exclusion chromatography methods, to yield a purified protein. This is especially important for the SHERLOCK assay, as any RNase contamination can give false positive results. The protein was then concentrated to 2 mg/mL, aliquoted and frozen at -80°C. The protein purification gel below displays the various stages of the expression and purification process. Thanks to the D40 protein gurus (Arthur, Alan and Zac, to name but a few) for all their help and advice during the purification.
Expression and purification of the Cas13a protein. (Left) The shake flasks used to grow the E. coli to express the Cas13a. (Right) SDS-PAGE protein purification gel displaying the various stages of the protein purification procedure, with protein purity increasing from left to right. (A more detailed figure is displayed at the end of this blog).
Designing crRNA target sequences
With the biosensor protein in hand, the team designed some genetic sequences that guide the protein to the target RNA, if it is present. Two computational biology tools were used: ADAPT [Metsky et al., (2022)] and BLASTN. Firstly, ADAPT was used to identify a target sequence unique to a particular organism. A house-keeping gene (rpoB) was used as a starting point, since it is commonly used as a molecular marker in microbial ecology studies [Case et al., (2017)]. Since our preliminary studies concerned the detection of a particular microorganism within mock microbial community DNA, we ran ADAPT against the rpoB sequences of other species in the community. The absence of the target sequence in other bacterial species was confirmed using BLASTN. The designed crRNA were ordered as DNA templates and then expressed using in vitro transcription, as per the SHERLOCK protocol [Kellner et al., (2019)].
Testing the biosensors
Mock microbial community DNA was used to validate the biosensor. crRNA sequences were designed and synthesised for all microorganisms found in the mock microbial community. Every design successfully gave a positive result (see below), detecting the target in the mock community sample, as intended. Furthermore, testing without the DNA present, and testing a target (bird flu) not present in the community, both gave a negative result. Some of the SHERLOCK reagents are particularly expensive and so after this first test, the assay was optimised to reduce reagent input, whilst maintaining adequate fluorescence from a positive result (results not shown). This reduced the fluorescent output of a positive result from the mock community from around 600,000 AU to around 300,000 AU.
Testing SHERLOCK biosensors against mock community DNA
The next step was to test the biosensors for cross-reaction using E. coli DNA. The results show some of the biosensors ‘light up’ in response to E. coli DNA, giving a false positive result (see below). This is despite being designed to avoid targeting other microbes in the mock microbial community. Whilst the strength of the response is weaker, testing further biosensor design iterations would be necessary to identify those that are specific to particular microorganisms.
Testing SHERLOCK biosensors against mock community and E. coli DNA
Finally, the team tested four of the biosensors against two DNA samples that had been extracted from soil. Three of the four biosensors gave a positive result for both samples (see below). The measured signal was weaker than detection of species in the mock community. This could be a result of the lower concentration of targets in the environmental DNA samples. Environmental microbiomes contain many thousands of microorganisms, which would make detection of specific species challenging. These results could be confirmed by DNA sequencing the environmental DNA samples.
Testing SHERLOCK biosensors against mock community environmental DNA (D2 & S2)
The six laws of open source drug discovery are equally relevant to open source synthetic biology research (image reproduced from Todd et al., with permission)
All considered, these initial results show that further optimisation is required to achieve the main goal of this ambitious project: to allow the detection of specific bacterial species in water-based samples. Whilst the biosensors detected the target species, they appeared to be susceptible to activation by RNA from other species, resulting in false positive results. This living planet is estimated to be home to one trillion microbial species, making it tricky, but not impossible, to design biosensors for the detection of particular microorganisms.
We are sharing these results to encourage a mindset of open source research, for which six guiding principles have been proposed [Todd, (2016)].
We would like to thank the SynBio CDT for the funding that facilitated this project, the D40 and Biocompute labs for the use of lab space and equipment, and Kathleen Sedgley for the help in setting up the project.
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Supplementary
Expression and purification of the Cas13a protein. SDS-PAGE protein purification gel displaying the various stages of the protein purification procedure, with protein purity increasing from left to right. Lanes are as follows: L – protein ladder, 1 – Cleared cell lysate post sonication, 2 – Streptactin resin post binding, 3 – Streptactin resin post cleavage, 4 – Flow-through post cleavage, 5 – Concentrated sample post IEC , 6 – Concentrated sample post SEC, 7 – Diluted Cas13a aliquot (5 µL), 8 – Diluted Cas13a aliquot (1 µL). The molecular weight of SUMO-Cas13a is 155 kDa and Cas13a (post SUMO cleavage) is 139 kDa.
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References:
Case, R. J. et al. (2007) “Use of 16S rRNA and rpoB Genes as Molecular Markers for Microbial Ecology Studies”, Applied and Environmental Microbiology, 73(1), pp. 278–288. Available at: doi.org/10.1128/AEM.01177-06.
Gootenberg, J. S. et al. (2017) “Nucleic acid detection with CRISPR-CAS13A/C2C2”, Science, 356(6336), pp. 438–442. Available at: doi.org/10.1126/science.aam9321.
Kellner, M. J. et al. (2019) “Sherlock: Nucleic acid detection with CRISPR nucleases”, Nature Protocols, 14(10), pp. 2986–3012. Available at: doi.org/10.1038/s41596-019-0210-2.
Metsky, H. C., et al. (2022). “Designing sensitive viral diagnostics with machine learning”, Nature Biotechnology, pp. 1–9. Available at: doi.org/10.1038/s41587-022-01213-5.
Todd, M. H. (2019). “Six Laws of open source drug discovery”, ChemMedChem, 14(21), pp. 1804–1809. Available at: doi.org/10.1002/cmdc.201900565.
Ben Hardy, a Research Associate in the School of Biochemistry at the University of Bristol, won a prize for his poster at the Synthetic Biology UK conference (7-8 November 2022).
Summarising work from his PhD, Ben’s poster (Computational Design of a de novo Transmembrane Cytochrome) highlights the successful design of an artificial protein capable of electron transport within membranes – such a protein is an essential component for constructing novel bioenergetic complexes within cells.
This was Ben’s second poster success this year – he won first prize at the Advances in Protein Folding, Evolution & Design conference (April 2022) for the poster: ‘Computational design of Bioenergetic Membrane Proteins’ (pictured).
Congratulations to 
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