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Latest revision as of 18:47, 9 July 2015
pTAL-TF
To show the functionality of our TAL protein as well as the impact of the VP 64 transcription factor fusion protein, we used a TAL-VP64 fusion construct targeting a minimal promotor coupled with the secreted alkaline phosphatase (SEAP). The product of the reporter gene SEAP is - as the name tells - a phosphatase that is secreted by the cells into the surrounding media. The existence of SEAP and therefore the activity of the promotor can be measured by the addition of para-Nitrophenylphosphate (pNPP). The SEAP enzyme catalyzes the reaction from pNPP to para-Nitrophenol, this new product absorbs light at 405 nm and can be measured via photometry. 
This reporter system gives us a couple of advantages over standard EGFP or luciferase systems. First of all, the SEAP is secreted into the cell culture media, therefore we don't have to lyse our cells for measuring, but just take a sample from the supernatant.
We are also able to measure one culture multiple times, e.g. at two different points in time. Another advantage is the measurement via photometry which makes the samples quantitively comparable. Interestingly, we did not have to clone a TALE binding site upstream of the minimal promoter (which would be required for other DNA binding proteins) but simply produced a TALE that specifically bound to the given sequence.
This reporter system gives us a couple of advantages over standard EGFP or luciferase systems. First of all, the SEAP is secreted into the cell culture media, therefore we don't have to lyse our cells for measuring, but just take a sample from the supernatant.
We are also able to measure one culture multiple times, e.g. at two different points in time. Another advantage is the measurement via photometry which makes the samples quantitively comparable. Interestingly, we did not have to clone a TALE binding site upstream of the minimal promoter (which would be required for other DNA binding proteins) but simply produced a TALE that specifically bound to the given sequence.
Usage and Biology
Gene activation
Experimental design
The experiment was done with four different transfections, either no plasmid, only the TAL vector, only the SEAP plasmid or a cotransfection of both plasmids. The cells were seeded on a twelve well plate the day before in 500µl culture media per well. The transfection was done with CaCl2.
Activation of transcription
To show that our TAL effectors are actually working, we used our completed toolkit to produce a TAL protein which is fused to a VP64 transcription factor. With this TAL-TF construct we targeted a sequence upstream of a minimal promotor that controls transcription of the enzyme secreted alkaline phosphatase (SEAP). In theory, the TAL domain should bring the fused VP64 domain in close proximity to the minimal promotor to activate the transcription of the repoter gene SEAP. The phosphatase is secreted an acummulates in the cell culture media. After 24 and 48 hours, we took samples from the media, stored them at -20°C, and subjected them to photometric analysis.
As it is observable in the graph, co-transfection of cells with TAL and SEAP plasmids(++) yielded a high increase in SEAP activity, compared to the control samples. Also the control experiment with a TAL-VP64 targeting a random sequence shows the specificity of our system. The graph shows the average value of three biological replicates with its standard deviation. We further performed a t-test (Table) to prove if our experiment is statistically significant. As it is clearly observable, the p-values range below a value of 0,05, which indicates that our TAL transcription factor is able to elevate the transcription of the SEAP gene in a statistically significant manner.
After addition of pNPP, the substrate of SEAP, the activity of SEAP was measured over time. In the next image, the results of the first nine minutes of this measurement are shown. After this time, the OD of the double transfection (++) rose too high to be measured by our photometer. As it is clearly visible, the sample with the double transfection shows a profound increase in the OD. This points to the fact that great amounts of SEAP have been secreted into the cell culture media due to elevated gene expression. In the other samples almost no SEAP activity was measureable. The sample transfected with only the SEAP plasmid showed the highest OD but this effect was not statistically significant (p-value:0,25/0,51).
In the samples that had been taken 48h after double transfection, the same effects could be demonstrated.
Furthermore, we reapeated the same experiment for a second time. The corresponding data can be viewed here:
As it is observable in the graph, co-transfection of cells with TAL and SEAP plasmids(++) yielded a high increase in SEAP activity, compared to the control samples. Also the control experiment with a TAL-VP64 targeting a random sequence shows the specificity of our system. The graph shows the average value of three biological replicates with its standard deviation. We further performed a t-test (Table) to prove if our experiment is statistically significant. As it is clearly observable, the p-values range below a value of 0,05, which indicates that our TAL transcription factor is able to elevate the transcription of the SEAP gene in a statistically significant manner.
After addition of pNPP, the substrate of SEAP, the activity of SEAP was measured over time. In the next image, the results of the first nine minutes of this measurement are shown. After this time, the OD of the double transfection (++) rose too high to be measured by our photometer. As it is clearly visible, the sample with the double transfection shows a profound increase in the OD. This points to the fact that great amounts of SEAP have been secreted into the cell culture media due to elevated gene expression. In the other samples almost no SEAP activity was measureable. The sample transfected with only the SEAP plasmid showed the highest OD but this effect was not statistically significant (p-value:0,25/0,51).
In the samples that had been taken 48h after double transfection, the same effects could be demonstrated.
Furthermore, we reapeated the same experiment for a second time. The corresponding data can be viewed here:
Second Experiment.
Sequence and Features
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 966
Illegal suffix found in sequence at 4039 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 966
Illegal SpeI site found at 4040
Illegal PstI site found at 4054
Illegal NotI site found at 972
Illegal NotI site found at 2146
Illegal NotI site found at 4047 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 966
Illegal BglII site found at 1563
Illegal BglII site found at 2914
Illegal BamHI site found at 2217
Illegal BamHI site found at 2920 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 966
Illegal suffix found in sequence at 4040 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 966
Illegal XbaI site found at 981
Illegal SpeI site found at 4040
Illegal PstI site found at 4054 - 1000COMPATIBLE WITH RFC[1000]