Wet-Lab : Parts Overview

Registry parts overview

Access our lab notebooks HERE!

Riboswitch (Adapted and improved from the iGEM Nottingham 2019 team)

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References

  1. 2.Inés C. Cañadas, Daphne Groothuis, Maria Zygouropoulou, Raquel Rodrigues, and Nigel P. Minton, ACS Synthetic Biology 2019 8 (6), 1379-1390, DOI: 10.1021/acssynbio.9b00075

  2. Heap, J. T., Pennington, O. J., Cartman, S. T. & Minton, N. P. A modular system for Clostridium shuttle plasmids. J. Microbiol. Methods 78, 79–85 (2009). 2. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. & Pease, L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61–8 (1989).

  3. Chambers, S. P., Prior, S. E., Barstow, D. A. & Minton, N. P. The pMTL nic− cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. Gene 68, 139–149 (1988).

  4. Mordaka, P. M. & Heap, J. T. Stringency of synthetic promoter sequences in Clostridium revealed and circumvented by tuning promoter library mutation rates. (2017).

  5. Topp, S. et al. Synthetic riboswitches that induce gene expression in diverse bacterial species. Appl. Environ. Microbiol. 76, 7881–4 (2010).

  6. Williams, D. R., Young, D. I. & Young, M. Conjugative plasmid transfer from Escherichia coli to Clostridium acetobutylicum. J. Gen. Microbiol. 136, 819–826 (1990).

  7. Schwarz, K. M. et al. Towards improved butanol production through targeted genetic modification of Clostridium pasteurianum. Eng. 40, 124–137 (2017).


GEM 1: Dual plasmid induced CRISPR/Cas9 editing tool for Clostridium genus probiotics

Best new basic part: We chose the codon optimized Cas9 plasmid to be nominated for the best basic part section due to its expandable/modular platform. Endogenous CRISPR/Cas systems have not been studied in a number of non-model gut bacteria. Simultaneously, as evidenced by the first engineering cycle Sp.Cas9 may lead to cell death in some bacteria, therefore this codon optimized Cas9 system has shown it’s efficiency in C. sporogenes and C. butyricum. Keeping in mind the bacteria of interest to create a template gRNA, this can be used in precision microbiome editing to engineer the desired probiotics for therapeutic application.

Design Considerations:

  • Codon usage - The codon usage of the native cas9 gene was analyzed and altered to match the preferred codons of genus Clostridium. This helps ensure efficient translation.

  • GC content - The GC content was adjusted to fit the typical range of Clostridium genes. Extreme GC content can negatively impact transcription and translation.

  • Restriction sites - Common restriction enzyme sites were avoided in the optimized sequence to facilitate future cloning efforts.

  • Repeats - Long repeats and inverted repeats were minimized to avoid unwanted secondary structures in the mRNA.

  • RBS - An optimal ribosome binding site was included upstream of the start codon to promote translation initiation.

  • mRNA structure - Potential mRNA secondary structures that could inhibit translation were analyzed and disrupted.

  • Toxic sequences - Known toxic motifs like endonuclease recognition sites were removed.

  • . Sequence continuity - The optimized sequence was designed to keep overall continuity relative to the native gene.

  • Synthesis - The optimized sequence parameters were set to enable successful gene synthesis by GenScript.

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References:

  1. Mamou Diallo, Rémi Hocq, Florent Collas, Gwladys Chartier, François Wasels, Hani Surya Wijaya, Marc W.T. Werten, Emil J.H. Wolbert, Servé W.M. Kengen, John van der Oost, Nicolas Lopes Ferreira, Ana M. López-Contreras,Adaptation and application of a two-plasmid inducible CRISPR-Cas9 system in Clostridium beijerinckii, Methods,Volume 172, 2020, Pages 51-60,ISSN 1046-2023,https://doi.org/10.1016/j.ymeth.2019.07.022.

  2. Wasels, F., Jean-Marie, J., Collas, F., López-Contreras, A. M., & Lopes Ferreira, N. (2017b). A two-plasmid inducible CRISPR/cas9 genome editing tool for clostridium acetobutylicum. Journal of Microbiological Methods, 140, 5–11. https://doi.org/10.1016/j.mimet.2017.06.010

  3. Wasels F, Chartier G, Hocq R, Lopes Ferreira N. A CRISPR/Anti-CRISPR Genome Editing Approach Underlines the Synergy of Butanol Dehydrogenases in Clostridium acetobutylicum DSM 792. Appl Environ Microbiol. 2020 Jun 17;86(13):e00408-20. doi: 10.1128/AEM.00408-20. PMID: 32385078; PMCID: PMC7301843.


Design of an toxin-antitoxin auxotrophy part for improved biosafety:

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This design was created for the biocontainment of engineered bacteria in the lab and as a supplementary control in engineered probiotics. We designed this after talking to professionals at the Synbiobeta conference who suggested auxotrophy to unnatural amino acids over designing kill switches.

Sensitivity to IY in this system is high and coupling it with an inducible riboswitch (BBa_K4943058) will prevent horizontal gene transfer and colonization in the gut.

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References

  1. Kato Y. An engineered bacterium auxotrophic for an unnatural amino acid: a novel biological containment system. PeerJ. 2015 Sep 15;3:e1247. doi: 10.7717/peerj.1247. PMID: 26401457; PMCID: PMC4579030.


Design of education part:

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  1. Shixiu Cui, Xueqin Lv, Yaokang Wu, Jianghua Li, Guocheng Du, Rodrigo Ledesma-Amaro, and Long Liu, ACS Synthetic Biology 2019 8 (8), 1826-1837, DOI: 10.1021/acssynbio.9b00140