Part:BBa_K4201006:Design

cytoTDS2-MBP

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 974
  Illegal PstI site found at 661
  Illegal PstI site found at 2222
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 661
  Illegal PstI site found at 2222
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 974
  Illegal PstI site found at 661
  Illegal PstI site found at 2222
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 974
  Illegal PstI site found at 661
  Illegal PstI site found at 2222
  Illegal AgeI site found at 810
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1
  Illegal BsaI.rc site found at 3566
  

Design Notes

Codon optimization for Glycine max (soybean) is a unique design consideration for all of CU Boulder’s coding sequences including our cytoTDS2-MBP. 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. Additionally, the chloroplast location tag on the 5’ end of the coding region for taxadiene synthase was removed. This modification forces the encoded protein to be placed into the cytosol. The design of the Maltose binding protein attached to the 3’ end of the TDS coding region 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 protein³. 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.

A diagram of the metabolic pathway for the synthesis of paclitaxel. Taxadiene synthase converts GGPP into taxadiene, as highlighted in red.

Source

This Taxadiene synthase-maltose binding protein sequence originates from Taxus chinensis var. mairei. All of our DNA fragments are obtained via de novo synthesis by iGEM sponsors Twist Bioscience and Integrated DNA technologies.

References

1. Zou, D. et al. Production of a novel lycopene-rich soybean food by fermentation with Bacillus amyloliquefaciens. LWT 153, 112551 (2022).
2. Xiong, X. et al. The Taxus genome provides insights into paclitaxel biosynthesis. Nat. Plants 7, 1026–1036 (2021).
3. Protein Chromatography: Methods and Protocols. vol. 1485 (Springer New York, 2017).