Part:BBa_K3570023
Bidirectional promoter C120
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 14
Illegal NotI site found at 441 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage
The last decade was particularly rich in new optogenetic systems approaches. Elected “Method of the year” by Nature readers in 2010, optogenetics had a huge wave of popularity. Optogenetics comprises different branches of application, and one of them aims at the precise regulation of gene expression level.
The need for precise, non-toxic, and homogenous effectors that would drive positively the level of expression of the gene of interest (GOI) led our choice to use the EL222 system. It consists of the effector protein, named EL222 (BBa_K3570021), and the associated promoter, named C120, put upstream to the gene of interest (fig. 1-2).
Our ultimate goal was to regulate the levels of expression of two genes simultaneously, so we created a bidirectional C120 promoter. When put next to two genes of interest, the bidirectional promoter allows controlling positively in-vivo gene expression level via the light-sensitive transcription factor EL222 (BBa_K3570021). Each of the two parts of the promoter contains 5 repeats of EL222-binding site and one repeat of a minimal promoter.
Biology
The EL222 is a light-sensitive transcription factor that englobes two functional domains. A light-oxygen-voltage-sensing (LOV) domain that responds to illumination at 450nm, which leads to a homodimerization of two entities of EL222. The second one is an HTH (helix-turn-helix)- DNA binding domain, that would recognize and bind specifically to C120 repeat. The cofactor used by this optogenetic system is FMN (flavin mononucleotide) EL222 protein originated from Erythrobacter litoralis. EL222 can be fused to a nuclear localisation site and to vp16 protein. The resulting protein can be used in yeast as transcription factor which activate the transcription of a gene whose promoter is C120.
Experiments
Team iGEM Toulouse 2020 did not have sufficient time to complete the cloning and hence, to test this part functionality.
References:
- Zhao, E. M., Zhang, Y., Mehl, J., Park, H., Lalwani, M. A., Toettcher, J. E., & Avalos, J. L. (2018). Optogenetic regulation of engineered cellular metabolism for microbial chemical production. Nature, 555(7698), 683–687.
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