Difference between revisions of "Part:BBa K3570005"

Latest revision as of 17:44, 26 October 2020

EL222 blue optogenetic regulation system in S. cerevisiae

Introduction

The purpose of this biobrick is to provide S. cerevisiae with the optogenetic gene expression regulation system. This is achieved by expressing a fusion protein NLS-EL222-VP16. The functionality can be directly verified by green fluorescent protein(GFP) fruorescence assay.This construction should be put into replicative or integrative plasmid.

Design

Fig. 1: EL222 construct. The gene coding for NLS-EL222-vp16 is between XbaI and BamHI restriction sites in order to be integrated into BBa_K3570003 or BBa_K3570001. We used the terminator ADH1 and the promoter pPGK1 for the gene NLS-EL222-vp16. The bidirectional promoter C120 (pC120) can be found here. We added the GFP after pC120 to test it before its integration in the yeast.

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 positive regulation of 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 machinery recruitment via VP16, and hence the transcription activation of the gene positioned downstream of the artificial promoter C120 (BBa_K3570023) (fig. 2-3).

The GOI here is GFP (fig. 1). The idea is to evaluate the optogenetic activation ratio, speed, and durability using GFP fluorescence. The GFP sequence was taken from BBa_E0040 part. PGK1 promoter sequence was identified from personal communication with Dr. Jean-Luc Parrou. ADH1 terminator sequence was identified from personal communication with Dr. Anthony Henras.

Fig. 2: Non-activated optogenetic system. GOI: gene of interest.

Fig. 3: Activated optogenetic system upon blue light illumination. GOI: gene of interest.

Experiments

Team iGEM Toulouse 2020 did not have sufficient time to complete the cloning and hence, to test this part functionality.

References

• [1]- 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
• [2]- 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
• [3]- 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