Coding

Part:BBa_K3570021

Designed by: Anton Mykhailiuk   Group: iGEM20_Toulouse_INSA-UPS   (2020-10-06)
Revision as of 06:53, 9 October 2020 by Brice (Talk | contribs)


NLS-VP16-EL222 photosensitive transcription factor

Usage

The development of biotechnology for the industry pushed it to increase the productivity and safety of the biotechnological processes. One of those breakthroughs were the inducible promoters. The principle is based on the recruitment of transcriptional regulators using small-molecule responsive DNA-binding proteins (Janicki et al., 2004). However, chemically-induced systems present many drawbacks such as an uncontrolled spatiotemporal resolution, the speed of the cellular uptake, and the release of the inducer.

In order to meet these requirements, it is possible to employ a clever optogenetic system that allows regulating the expression of the gene of interest (GOI). Previously described photosensitive transcription factor (TF) that consists of a nuclear localization signal (NLS), the VP16 transactivation domain, and the bacterially-derived protein EL222 (Motta-Mena et al.., 2014; Nash et al., 2011). EL222 has the capacity to self-dimerize upon light stimulation thanks to its light oxygen and voltage (LOV) domain. EL222 has a DNA binding domain and the artificial promoter C120 contains 5 repeated binding sites for the fixation of EL222 and one sequence of a minimal promoter. When exposed to blue light (450nm), this system allows the transcription activation of the gene positioned downstream of the artificial promoter C120 (<a href="https://parts.igem.org/Part:BBa_K3570023">BBa_K3570023</a>) (fig. 1-2).

Fig. 1: Non-activated optogenetic system. GOI: gene of interest.
Fig. 1: Activated optogenetic system upon blue light illumination. GOI: gene of interest.
..

References

  • Janicki, S. M., Tsukamoto, T., Salghetti, S. E., Tansey, W. P., Sachidanandam, R., Prasanth, K. V., Ried, T., Shav-Tal, Y., Bertrand, E., Singer, R. H., & Spector, D. L. (2004). From Silencing to Gene Expression. Cell, 116(5), 683–698. https://doi.org/10.1016/s0092-8674(04)00171-0
  • Motta-Mena, L. B., Reade, A., Mallory, M. J., Glantz, S., Weiner, O. D., Lynch, K. W., & Gardner, K. H. (2014). An optogenetic gene expression system with rapid activation and deactivation kinetics. Nature Chemical Biology, 10(3), 196–202. https://doi.org/10.1038/nchembio.1430
  • Nash, A. I., McNulty, R., Shillito, M. E., Swartz, T. E., Bogomolni, R. A., Luecke, H., & Gardner, K. H. (2011). Structural basis of photosensitivity in a bacterial light-oxygen-voltage/helix-turn-helix (LOV-HTH) DNA-binding protein. Proceedings of the National Academy of Sciences, 108(23), 9449–9454. https://doi.org/10.1073/pnas.1100262108


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 256
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 256
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 256
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 256
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 256
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
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