Difference between revisions of "Part:BBa K2483008"
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<partinfo>BBa_K2483008 short</partinfo> | <partinfo>BBa_K2483008 short</partinfo> | ||
| − | This is the final construct | + | 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. | + | 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 | + | 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. | + | 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. | 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. | ||
Latest revision as of 11:39, 1 November 2017
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
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 13
- 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 13
- 21INCOMPATIBLE 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 - 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 13
- 25INCOMPATIBLE 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 - 1000INCOMPATIBLE 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