Difference between revisions of "Part:BBa K801042"

(Characterisation via Luciferase Assay)
(Characterisation via Luciferase Assay)
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[[File:TUM12_GAL4_LSPS.png|thumb|right|300px|'''Fig. 7:''' Evaluation of Luciferase Assay for the GAL4 based system.]]
 
[[File:TUM12_GAL4_LSPS.png|thumb|right|300px|'''Fig. 7:''' Evaluation of Luciferase Assay for the GAL4 based system.]]
  
PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is quite low.
+
PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is close to zero.
  
As expression the promoter system was driven by the quite strong TEF2 promoter and was transfected on a high copy vector, there probably was a quite high level of both fusion proteins which led to unspecific binding and a quite high leaky transcription rate for the dark and low intensity samples. This problem could be dealt with by using a weaker promoter or a low copy vector.
+
As expression the promoter system was driven by the quite strong pTEF1 promoter and was transfected on a high copy vector, there probably was a quite high level of both fusion proteins which led to unspecific binding and a quite high leaky transcription rate for the dark and low intensity samples. This problem could be dealt with by using a weaker promoter or a low copy vector.
  
 
The high intensity sample still shows a 3 fold increased induction for a 10 fold increased light intensity compared to the low intensity.
 
The high intensity sample still shows a 3 fold increased induction for a 10 fold increased light intensity compared to the low intensity.

Revision as of 01:07, 27 October 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 (Pfr-form); the Pfr 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 Pfr form (red light exposition) or back to its ground-state Pr (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.

One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.

If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.

LexA based light-switchable-promoter system

For more information about the LexA based system, please see here: BBa_K801043

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.

Characterisation via Luciferase Assay

Fig. 7: Evaluation of Luciferase Assay for the GAL4 based system.

PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is close to zero.

As expression the promoter system was driven by the quite strong pTEF1 promoter and was transfected on a high copy vector, there probably was a quite high level of both fusion proteins which led to unspecific binding and a quite high leaky transcription rate for the dark and low intensity samples. This problem could be dealt with by using a weaker promoter or a low copy vector.

The high intensity sample still shows a 3 fold increased induction for a 10 fold increased light intensity compared to the low intensity.

References


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 862
    Illegal BglII site found at 2795
    Illegal BamHI site found at 2877
    Illegal XhoI site found at 2828
    Illegal XhoI site found at 2847
    Illegal XhoI site found at 4982
    Illegal XhoI site found at 5316
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2240
    Illegal AgeI site found at 5540
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 4367
    Illegal BsaI site found at 4773
    Illegal BsaI site found at 5235
    Illegal BsaI.rc site found at 205
    Illegal BsaI.rc site found at 1986
    Illegal SapI site found at 3044