Difference between revisions of "Part:BBa K4201019:Design"

Latest revision as of 10:51, 12 October 2022

Design Notes

Codon optimization for Glycine max (soybean) is a unique design consideration for all of CU Boulder’s coding sequences including our crtE-cytoTDS-MBP_T5αOH_RUBY construct. Codon optimization is the intentional use of specific codons for specific amino acids, dependent on what tRNAs are most abundant in the organism. While codon optimization is a common consideration for synthetic biologists, our sequences are unique for iGEM because they are intended for expression in soybeans.

The designed construct of CrtE-cytoTDS2-MBP encodes for one long protein in order to determine if this modification benefits the channeling of substrates from the CrtE to the taxadiene synthase, resulting in higher yields than an uncombined protein. The design of the Maltose binding protein attached to the 3’ end of the TDS coding region of the crtE-cytoTDS-MBP part is intended to assist with protein solubility in the cytosol. Maltose binding protein is known as a “fusion partner,” which is used for producing recombinant proteins in bacterial cells. It also has a high rate of translation, and ensures the proper folding and solubility of the target protein⁵.

Gmubi is constitutive promoter native to Glyine max⁶, while AtHSP is the terminator of a heat shock protein that has shown to promote expression in plants⁷.

This assembly was made using NEB 10-beta cells and GoldBio EHA105 agrobacterium as intermediate hosts. Parts BBa_K4201013 (Amp BsaI) and BBa_K4201014 (Chlor AaRI) were used as intermediate backbones for Golden Gate assembly. The construct was integrated into part BBa_K4201015 (Kan BsaI) as a final backbone before transfection into Glycine max.

Source

CrtE is a GGPP synthase from Pantoea ananatis LMG 20103¹.

Gmubi promoters originate from Glycine max ⁶.

AtHSP terminators originate from the Arabidopsis thaliana genome⁷.

cytoTDS2 is a taxadiene synthase native to Taxus chinensis var. mairei optimized for use in Glycine max².

T5αOH is a hydroxylase from Taxus baccata that converts taxadiene into taxadiene-5α-ol, a step in the paclitaxel pathway³.

RUBY is a reporter gene from the order Caryophyllales⁴.

MBP is a protein that contributes to stability during protein transport⁵.

De novo Synthesis was completed by iGem sponsors IDT and Twist Biosciences.

References

1. Majer, E., Llorente, B., Rodríguez-Concepción, M. & Daròs, J.-A. Rewiring carotenoid biosynthesis in plants using a viral vector. Sci. Rep. 7, 41645 (2017).

2. Xiong, X. et al. The Taxus genome provides insights into paclitaxel biosynthesis. Nat. Plants 7, 1026–1036 (2021).

3. Nagaya, S., Kawamura, K., Shinmyo, A. & Kato, K. The HSP Terminator of Arabidopsis thaliana Increases Gene Expression in Plant Cells. Plant Cell Physiol. 51, 328–32 (2010).

4. He, Y., Zhang, T., Sun, H., Zhan, H. & Zhao, Y. A reporter for noninvasively monitoring gene expression and plant transformation. Hortic. Res. 7, 1–6 (2020).

5. Lebendiker, M. & Danieli, T. Purification of Proteins Fused to Maltose-Binding Protein. Methods Mol. Biol. Clifton NJ 1485, 257–273 (2017).

6. De La Torre, C. M. & Finer, J. J. The intron and 5’ distal region of the soybean Gmubi promoter contribute to very high levels of gene expression in transiently and stably transformed tissues. Plant Cell Rep. 34, 111–120 (2015).

7. Nagaya, S., Kawamura, K., Shinmyo, A. & Kato, K. The HSP Terminator of Arabidopsis thaliana Increases Gene Expression in Plant Cells. Plant Cell Physiol. 51, 328–32 (2010).