Genetic regulation
Workpackage C: New mechanisms for regulation of gene expression
RNA-based mechanisms for gene regulation complement conventional transcriptional regulation, and can be highly flexible and modular. These mechanisms are common in nature, and are only now being harnessed for synthetic systems. OpenPlant aims to expand the availability of genetic regulatory elements across a range of plants, algae and cyanobacteria through projects on riboswitches, riboregulator circuits, cyanobacteria circuits and circadian rhythm regulators.
MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii
Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii, Crozet et al. (2018).
Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology. (Abstract from Crozet et al. (2018))
miRNA-Mediated Regulation of Synthetic Gene Circuits
Construction of a synthetic circuit to measure miRNA-dependent gene repression (Navarro and Baulcombe (2019), Figure 1A).
MicroRNAs (miRNAs), small RNA molecules of 20-24 nts, have many features that make them useful tools for gene expression regulation-small size, flexible design, target predictability, and action at a late stage of the gene expression pipeline. In addition, their role in fine-tuning gene expression can be harnessed to increase robustness of synthetic gene networks. We apply a synthetic biology approach to characterize miRNA-mediated gene expression regulation in the unicellular green alga Chlamydomonas reinhardtii. This characterization is then used to build miRNA-based tools that can facilitate the engineering of gene expression for new applications and improved traits in this alga. (Adapted from abstract Navarro and Baulcombe (2019))
Riboswitches
Development of Novel Riboswitches for Synthetic Biology in the Green Alga Chlamydomonas, Mershahi et al. (2020).
Riboswitches are RNA regulatory elements that bind specific ligands to control gene expression. Because of their modular composition, where a ligand-sensing aptamer domain is combined with an expression platform, riboswitches offer unique tools for synthetic biology applications. The simplicity of riboswitch incorporation in current design platforms will facilitate the generation of genetic circuits to advance synthetic biology and metabolic engineering of microalgae. (Adapted from abstract Mershahi et al (2020))
Publications
Mehrshahi P., Nguyen G.T.D.T., Gorchs Rovira A., Sayer A., Llavero-Pasquina M., Huei Sin M.L., Medcalf E.J., Mendoza-Ochoa G.I., Scaife M.A., and Smith A.G. (2020) Development of Novel Riboswitches for Synthetic Biology in the Green Alga Chlamydomonas. ACS Synth. Biol. 9 (6) 1406–1417. doi: 10.1021/acssynbio.0c00082
Andriotis VME, Smith AM (2019) The plastidial pentose phosphate pathway is essential for postglobular embryo development in Arabidopsis. PNAS 116 (30) 15297-15306; DOI: 10.1073/pnas.1908556116
Rovira, A.G. and Smith, A.G. (2019), PPR proteins – orchestrators of organelle RNA metabolism. Physiol Plantarum 166: 451-459. https://doi.org/10.1111/ppl.12950
Chung, B.Y.-W., Valli, A., Deery, M.J., Navarro, F.J., Brown, K., Hnatova, S., Howard, J., Molnar, A., and Baulcombe, D.C. (2019). Distinct roles of Argonaute in the green alga Chlamydomonas reveal evolutionary conserved mode of miRNA-mediated gene expression. Sci. Rep. 9: 1–12. doi: 10.1038/s41598-019-47415-x
Navarro FJ and Baulcombe D. (2019) miRNA-mediated regulation of synthetic gene circuits in the green alga Chlamydomonas reinhardtii. ACS Synth Biol. 8(2):358-370. doi: 10.1021/acssynbio.8b00393.
Crozet P, Navarro FJ, Willmund F, Mehrshahi P, Bakowski K, Lauersen KJ, Pérez-Pérez ME, Auroy P, Gorchs Rovira A, Sauret-Gueto S, Niemeyer J, Spaniol B, Theis J, Trösch R, Westrich LD, Vavitsas K, Baier T, Hübner W, de Carpentier F, Cassarini M, Danon A, Henri J, Marchand CH, de Mia M, Sarkissian K, Baulcombe DC, Peltier G, Crespo JL, Kruse O, Jensen PE, Schroda M, Smith AG, Lemaire SD (2018) Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synth Biol. 7(9):2074-2086. doi: 10.1021/acssynbio.8b00251.
Brodie J, Chan CX, De Clerck O, Cock JM, Coelho SM, Gachon C, Grossman AR, Mock T, Raven JA, Smith AG, Yoon HS, Bhattacharya D (2017). The Algal Revolution. Trends Plant Sci. 22(8):726-738. doi: 10.1016/j.tplants.2017.05.005.
Moses T, Mehrshahi P, Smith AG, Goossens A (2017) Synthetic biology approaches for the production of plant metabolites in unicellular organisms. J Exp Bot, 68: 4057-4074. doi: 10.1093/jxb/erx119
Nguyen GTDT, Scaife MA, Helliwell KE, Smith AG (2016) Role of riboswitches in gene regulation and their potential for algal biotechnology. J Phycol. 52(3):320-8. doi: 10.1111/jpy.12416.
Scaife MA, Smith AG (2016) Towards developing algal synthetic biology. Biochem Soc Trans. 44(3):716-22. doi: 10.1042/BST20160061.
Scaife MA, Nguyen GTDT, Rico J, Lambert D, Helliwell KE, Smith AG (2015) Establishing Chlamydomonas reinhardtii as an industrial biotechnology host. Plant J. 82(3):532-46. doi: 10.1111/tpj.12781.