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Team:Freiburg/Project/Experiments
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<div align="justify">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 targetting a minimal promotor coupled with the secreated 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 surrunding 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 formed product absorps light at 405 nm and can be measured via photometer. <img src="http://imageshack.us/a/img189/6140/seapplasmid.png" align="right" padding:0px width="450px" hspace="20"/> | <div align="justify">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 targetting a minimal promotor coupled with the secreated 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 surrunding 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 formed product absorps light at 405 nm and can be measured via photometer. <img src="http://imageshack.us/a/img189/6140/seapplasmid.png" align="right" padding:0px width="450px" hspace="20"/> | ||
| - | This reporter system gives us a couple of advantages over standard egfp or luciferase systems. First of all the SEAP is secreated into the cell culture media, | + | This reporter system gives us a couple of advantages over standard egfp or luciferase systems. First of all the SEAP is secreated into the cell culture media, therefore we dont have to destroy our cells to measure, but just take a sample from the supernatant. We are also able to measure one culture multiple times, e.g. at two different time points. Another advantage is the measurement via photometer which makes the samples quantitive compareable. |
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Revision as of 16:55, 26 September 2012
Experiments
Gene activation
This reporter system gives us a couple of advantages over standard egfp or luciferase systems. First of all the SEAP is secreated into the cell culture media, therefore we dont have to destroy our cells to measure, but just take a sample from the supernatant. We are also able to measure one culture multiple times, e.g. at two different time points. Another advantage is the measurement via photometer which makes the samples quantitive compareable.
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 after a cellculture course protocol written by the lab of professor Weber.
Results
The toolkit
The creation of a toolkit with 96 different parts not only means a lot of labwork but also a lot of organisational tasks, sequencing and analysis. We don't want to bore you with the 96 sequences of our finished biobricks, but we want to give you one example of a finished biobrick and highlight some of the interesting and important strips in its sequence. If you are interested in the other sequences, just have a look at our parts section or go to the [http://partsregistry.org Registry of Standard Biological Parts].
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In this sequence of our biobrick AA1, the main features of all our biobricks are highlighted. As pointed out in the Golden Gate Standard section of our project description, all direpeat plasmids are submitted in the Golden Gate Standard, that was developed by us and which is fully compatible with existing iGEM standards. In yellow you can see the direpeat gene fragment itself, the green parts are iGEM restriction sites (a requirement for all biobricks), the sequence written in red is part of the psb1C3 vector, the blue sequences are recognition sites for BsmB1 and the red boxes are the cutting sites of BsmB1.
Creation of TAL sequences - Golden Gate Cloning
Admittedly, our GATE assembly kit is a little larger than the kit published from the Zhang kit in Nature this year (it comprises 78 parts). But considering that future iGEM teams can easily combine the parts to form more than 67 million different effectors, we believe that it was worth the effort. Now, to get from the toolbox to the finished TAL effector, you only need a few components: six direpeats, one effector backbone plasmid, two enzymes and one buffer. If you mix these components and incubate in your thermocycler for 2.5 hours, you get your custom TAL effector. To put this in perspective: The average turnaround time for TALE construction with conventional kits is about two weeks! In the following sections, we want to show you the results of our trials with the finished biobricks and the sequencing of built TAL effector constructs.
Activation of transcription
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 secreated an acummulates in the cell culture media. After 24 and 48 hours we took samples from the media, kept them at -20°C, and subjected them two days later to photometric analysis. (For detailed experimental design and theory of SEAP look at our "Experiment" page in the project section.)
As it is observable in the graph, we were able to demonstrate a high increase in SEAP activity when co-transfecting TAL and SEAP plasmids (++), compared to the control samples. The graph shows the average value of three biological replicates with its standard deviation. We further performed a ttest (Table) to prove if our experiment is statistical significant. The yellow highlighted fields are the p-values for our double transfections. 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 statistical significant manner.
The next image shows the SEAP measurement over the first nine minutes, after this time the OD of the double transfection (++) got to high to be measured by our photometer. As you can see the OD of the double transfection rose very fast indicating a high amount of SEAP reacting with the substrate pNPP. In the other samples no SEAP was measureable, the sample transfected with only the SEAP plasmid showed the highest OD but not statisticaly significant (p-value:0,25/0,51). The results for the samples taken after 48 hours showed the same behavior. Also we reapeated the same experiment a second time. you can find the data here:
