Difference between revisions of "Part:BBa K4201021:Design"

Latest revision as of 10:51, 12 October 2022

CrtE_cytoTDS2-MBP_T5αOH_RUBY

Design Notes

Part information and design consideration can be found on respective basic parts pages.

This construct differs from similar composite parts like BBa_K4201019 by having different promoter (Gmubi) and terminator (AtHSP) amounts and locations. In these constructs, dashes(-) represent locations where genes were synthesized de novo. An underscore (_) symbolizes a location where we adhered genes together via Golden Gate assembly. Utilizing Golden Gate assembly allowed us to insert promoter and terminator sequences between genes of interest, and consequently enables the study of transcription efficacy based on promoter and terminator location.

RUBY, which is added to the end of this assembly, is a reporter gene that signifies if gene transformation was successful in Glycine max⁴.

Compared to BBa_K4201020, this construct contains the coding sequence for a maltose binding protein (MBP), which helps maintain protein stability during transport⁵.

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 max⁶.

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).