Part:BBa_K2483008

IAA producing genes fused to droplet forming Ddx4 for S.cerevisiae

This is the final construct of 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. Additionnally, we fused those to the N- and C-terminal domain of the protein Ddx4. Ddx4 is known to induce LLPS.

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 to compare the output of our enzymes with and without LLPS. The other construct for this experiment is BBa_K2483009: https://parts.igem.org/Part:BBa_K2483009.

We could verify before, that Ddx4 is capable of inducing LLPS in yeast cells (before this only done in HeLa cells).

As a promoter, we used GAL1 (strong, galactose-induced promoter) and the same 10xSG linker as in BBa_K2483007 (https://parts.igem.org/Part:BBa_K2483007).

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 13
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 13
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1089
  Illegal BamHI site found at 1302
  Illegal BamHI site found at 4585
  Illegal BamHI site found at 5989
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 13
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 13
  Illegal NgoMIV site found at 5032
  Illegal AgeI site found at 89
  Illegal AgeI site found at 3409
  Illegal AgeI site found at 5328
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 712
  Illegal BsaI site found at 2110
  Illegal BsaI site found at 5331
  Illegal BsaI.rc site found at 665