Part:BBa_K2483009

IAAM and IAAH yeast optimized under GAL1 control

This part was used as a control for our liquid-liquid phase separation (LLPS) project. We used the CDS of the Indoleacetic acid-producing enzymes IAAM and IAAH from the part BBa_K515100 (https://parts.igem.org/Part:BBa_K515100) by Imperial College 2011 and codon optimized them for S.cerevisiae.

Liquid-liquid phase separation occurs in all cells and can be imagined like oil droplets forming in water but here proteins are forming the droplets. We used this construct in yeast as a control to compare the output of our enzymes with and without LLPS. The other construct for this experiment is BBa_K2483008 (https://parts.igem.org/Part:BBa_K2483008).

As a promoter, we used GAL1 (strong, galactose-induced promoter) and CYC1 as a terminator.

The CDS' are facing away from each other because of the way this part was assembled (we used Gibson Assembly) and the original structure of the plasmid (pYES2/CT). That's why there's also a T7 promoter (which we didn't use though).

During the planning phase, we did not check for illegal restrictions sites. That's why we oversaw the SpeI site in the middle of the part.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 2301
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 2301
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 2301
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 2301
  Illegal NgoMIV site found at 1212
  Illegal AgeI site found at 916
  Illegal AgeI site found at 2086
  Illegal AgeI site found at 2377
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 3594
  Illegal BsaI.rc site found at 913