Difference between revisions of "Part:BBa K4729707"

Revision as of 17:12, 10 October 2023

Ptac + VirG TiBo542 N80D + repABCa

Introduction

Agrobacterium mediated transformation is one of the most prolific methods for plant engineering, by far the most common method for iGEM teams as well. However, working with plant Synbio is still far from a straightforward endeavor. This year, we set our sights on creating molecular tools that facilitate achieving successful transformation in non-model plant species, one of them is our Best Composite Part.

In nature, members of the genus Agrobacterium (Aphaproteobacteria) are soil-borne plant pathogens that integrate hormone producing genes in the host plant genome to cause growth deformities and tumors. This mechanism can be explored by substituting the oncogenes by genes of interest, thus integrating them in the plant chromosomes. The most well known strain, Agrobacterium tumefaciens, promotes crown-gall disease in most dicotyledonous plants, however, other strains such as Agrobacterium rhizogenes are also able to transform plant cells (Bahramnejad et al., 2019^[2]; Barton et al., 2018). Despite their wide use, Agrobacterium mediated plant transformation is still only well established for a handful of model organisms, which makes it hard for iGEM teams to engineer local species.

Part of the problem lies in the high specificity between plants and the necessary Agrobacterium strain for transformation. As we found out, getting hold of the correct Agrobacterium strain for a plant species (if one is even known) is extremely hard, reaching up to hundreds of Euros and months of shipping time. This is made even worse by the confusing and often conflicting nomenclature of existing strains (De Saeger et al., 2021).

Therefore, we created a composite part that aims to improve the efficiency and host range of Agrobacterium strains by leveraging the virG transcription factor as a Master-Switch plant transformation. With it, we hope to enable future teams to further extend the garden of plant projects in iGEM.

The molecular machinery of plant transformation

The Problem

The virG Master-Switch construct

Constitutive expression

Inducible expression

Sodium Salicylate inhibits cell growth

Cross-talk assay

Sequence and Features

References

[1] Anand, A., Che, P., Wu, E., & Jones, T. J. (2019). Novel Ternary Vectors for Efficient Sorghum Transformation. In Z.-Y. Zhao & J. Dahlberg (Eds.), Sorghum (Vol. 1931, pp. 185–196). Springer New York. https://doi.org/10.1007/978-1-4939-9039-9_13

[2] Bahramnejad, B., Naji, M., Bose, R., & Jha, S. (2019). A critical review on use of Agrobacterium rhizogenes and their associated binary vectors for plant transformation. Biotechnology Advances, 37(7), 107405. https://doi.org/10.1016/j.biotechadv.2019.06.004

[3] Barton, I. S., Fuqua, C., & Platt, T. G. (2018). Ecological and evolutionary dynamics of a model facultative pathogen: Agrobacterium and crown gall disease of plants. Environmental Microbiology, 20(1), 16–29. https://doi.org/10.1111/1462-2920.13976

[4] Bouchez, D., & Tourneur, J. (1991). Organization of the agropine synthesis region of the T-DNA of the Ri plasmid from Agrobacterium rhizogenes. Plasmid, 25(1), 27–39. https://doi.org/10.1016/0147-619X(91)90004-G

[5] Cho, H., & Winans, S. C. (2005). VirA and VirG activate the Ti plasmid repABC operon, elevating plasmid copy number in response to wound-released chemical signals. Proceedings of the National Academy of Sciences, 102(41), 14843–14848. https://doi.org/10.1073/pnas.0503458102

[6] Colognori, D., Trinidad, M., & Doudna, J. A. (2023). Precise transcript targeting by CRISPR-Csm complexes. Nature Biotechnology, 1–9. https://doi.org/10.1038/s41587-022-01649-9

[7] De Saeger, J., Park, J., Chung, H. S., Hernalsteens, J.-P., Van Lijsebettens, M., Inzé, D., Van Montagu, M., & Depuydt, S. (2021). Agrobacterium strains and strain improvement: Present and outlook. Biotechnology Advances, 53, 107677. https://doi.org/10.1016/j.biotechadv.2020.107677

[8] Gelvin, S. B. (Ed.). (2018). Agrobacterium Biology: From Basic Science to Biotechnology (Vol. 418). Springer International Publishing. https://doi.org/10.1007/978-3-030-03257-9

[9] Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J., & Voigt, C. A. (2019). Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nature Chemical Biology, 15(2), Article 2. https://doi.org/10.1038/s41589-018-0168-3

[10] Mostafavi, M., Lewis, J. C., Saini, T., Bustamante, J. A., Gao, I. T., Tran, T. T., King, S. N., Huang, Z., & Chen, J. C. (2014). Analysis of a taurine-dependent promoter in Sinorhizobium meliloti that offers tight modulation of gene expression. BMC Microbiology, 14, 295. https://doi.org/10.1186/s12866-014-0295-2

[11] Ozyigit, I. I., Dogan, I., & Artam Tarhan, E. (2013). Agrobacterium rhizogenes-Mediated Transformation and Its Biotechnological Applications in Crops. In K. R. Hakeem, P. Ahmad, & M. Ozturk (Eds.), Crop Improvement: New Approaches and Modern Techniques (pp. 1–48). Springer US. https://doi.org/10.1007/978-1-4614-7028-1_1

[12] Slightom, J. L., Durand-Tardif, M., Jouanin, L., & Tepfer, D. (1986). Nucleotide sequence analysis of TL-DNA of Agrobacterium rhizogenes agropine type plasmid. Identification of open reading frames. Journal of Biological Chemistry, 261(1), 108–121. https://doi.org/10.1016/S0021-9258(17)42439-2

[13] Stukenberg, D., Hensel, T., Hoff, J., Daniel, B., Inckemann, R., Tedeschi, J. N., Nousch, F., & Fritz, G. (2021). The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications in Vibrio natriegens. ACS Synthetic Biology, 10(8), 1904–1919. https://doi.org/10.1021/acssynbio.1c00126