Part:BBa_K2423007:Design

CsADH2946 under control with BBa_J04500

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 360
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 767
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1284
  Illegal SapI.rc site found at 392
  

Source

The original sequenced used for this BioBrick was discovered in a study from 2017 (1), where they analyzed the proteome of Crocus Sativus. From that they found the mRNA sequence, which was then reverse transcribed to cDNA. This was deposited on GenBank with accession number KU577903.

Design Notes

This part was designed by using the sequence from KU577903 (2) and codon optimzied using IDT's tool for Escherichia Coli. We generated new sequences until there were no EcoRI, XBaI, SpeI and PstI restriction sites. BBa_J04500 was included because we wanted to be able to induce the production of protein with IPTG. Thus, we would be able to overexpress our protein for purification. To be able to purify a his-tag was added to be able to immobilized affinity chromoatography (IMAC). Where the his-tag was placed was based on the homology models we made (3).

References

1. Gómez-Gómez L, Parra-Vega V, Rivas-Sendra A, Seguí-Simarro JM, Molina RV, Pallotti C, et al. Unraveling Massive Crocins Transport and Accumulation through Proteome and Microscopy Tools during the Development of Saffron Stigma. Int J Mol Sci [Internet]. 2017 Jan 1;18(1). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297711/

2. Crocus sativus isolate CsADH-2946 aldehyde dehydrogenase mRNA, complete cds. 2017 Feb 12; Available from: http://www.ncbi.nlm.nih.gov/nuccore/KU577903.1

3. Modeling iGEM Uppsala 2017; Available from: http://2017.igem.org/Team:Uppsala/Model