Latest revision as of 12:29, 12 October 2023

LexA based yeast light-swithable promoter system

composite part of Bba_K319003, K801039, BBa_K801011 , Bba_K319003, Bba_K801041, and BBa_K801011

Contribution of OUC-China

Group: iGEM Team OUC-China 2023

Author: Qingyu Wu

Summary: Red light optimization

Due to the fact that the fermentation of MEL-producing sap is thicker, it may cause the red light system to not work well. We therefore used a simulation based on Rombauer's law to model the propagation of light through the sap during fermentation to determine what lighting arrangement scheme we could use to maximize fermentation with minimal energy consumption in order to reduce the expense of subsequent experiments and increase their success.

I = I0 * e^(-αx)

The fermentation tank we used is a cylindrical vessel with a radius of approximately 10cm and a height of approximately 40cm. Considering the actual fermentation process, most of the fermentation culture is located in the upper layer, while the lower layer is mainly occupied by the product MEL. After continuous adjustment of the number and positioning of the lights, we finally determined that wrapping 8 light bulbs with an intensity of 180 umol/m^2/nm at distances of 4cm, 12cm, and 20cm from the top of the tank would ensure that the upper 2/3 region within the fermentation tank reaches a light intensity of 50 umol/m^2/nm.

Background and principles

This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e. g. LacZ or an auxotrophic marker.

Reverse yeast-two hybrid based light-switchable promoter system

This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photoconvertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).

Principle of light-dependent switching of gene-expression.

This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P_fr-form); the P_fr form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P_fr form (red light exposition) or back to its ground-state P_r (far-red light exposition or darkness).

GAL4 based light-switchable promoter system

For more information about the GAL4 based system, please see here: BBa_K801042

LexA based light-switchable-promoter system

In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signalling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.

In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e. g. BBa_K165031.

In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.

Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homolog protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the Arabidopsis chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.

References

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• http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009 Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. PLoS Genet, 5(1):e1000352.
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• http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985 Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of Escherichia coli K-12. J Bacteriol, 163(1):376–84.