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

Revision as of 09:06, 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 protein(2).

Source

CrtE is a GGPP synthase from Pantoea ananatis LMG 20103

RUBY is a reporter gene from the order Caryophyllales(1).

Gmubi promoters originate from Glycine max.

AtHSP terminators originate from the Arabidopsis thaliana genome.

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

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

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

References