Difference between revisions of "Part:BBa K801042"

Revision as of 23:47, 26 September 2012

GAL4 based yeast light-switchable promoter system

composite part of Bba_K319003, BBa_K801039, BBa_K801011 Bba_K319003, Bba_K801040, and BBa_K801011

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

In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part (BBa_K801040 and the second one is PIF3 fused to GAL4AD for the transcriptional activating part (BBa_K801039). This system allows us to control spatio-temporally the expression of our genes coded on pTUM104 and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 S. cerevisiae strain, which has an GAL4/GAL80 deletion.

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.

Biosynthesis of phycocyanobilin

Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P_fr is ~30 min, so continuous red light exposition is not nescessary. A great adventage is that light-sensitive odorant and flavorings will not be destroyed. Once phycocyanobilin is not naturally avaible in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalized by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.

Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).

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.

Sequence and Features