Part:BBa_K4201004:Design

CrtE-cytoTDS-MBP

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 55
  Illegal PstI site found at 3853
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 55
  Illegal PstI site found at 3853
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 671
  Illegal BglII site found at 2209
  Illegal BglII site found at 3256
  Illegal BamHI site found at 4307
  Illegal XhoI site found at 1632
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 55
  Illegal PstI site found at 3853
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 55
  Illegal PstI site found at 3853
  Illegal AgeI site found at 1374
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1
  Illegal BsaI.rc site found at 4487
  Illegal SapI.rc site found at 2430
  Illegal SapI.rc site found at 2636
  

Design Notes

Codon optimization for Glycine max (soybean) is a unique design consideration for all of CU Boulder’s coding sequences including our CrtE. 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 protein². 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 paclitacel. CrtE converts DMAPP into GGPP, outlined in blue. Taxadiene synthase converts GGPP into taxadiene, outlined in red.

Source

This mutant CrtE-Taxadiene Synthase-Maltose Binding Protein sequence originates from both Pantoea ananatis LMG 20103 and Taxus brevifolia. All of our DNA fragments are obtained via de novo synthesis by iGEM sponsors Twist Bioscience and Integrated DNA technologies.

References

1. Mutanda, I., Li, J., Xu, F. & Wang, Y. Recent Advances in Metabolic Engineering, Protein Engineering, and Transcriptome-Guided Insights Toward Synthetic Production of Taxol. Front. Bioeng. Biotechnol. 9, 632269 (2021).
2. Protein Chromatography: Methods and Protocols. vol. 1485 (Springer New York, 2017).