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Team:Freiburg/Project/Experiments
From 2012.igem.org
Gene activation
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 SEAo 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. 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, therfore we dont have to destroy our cells to meaure but just take a sample from the supernatant. Also we are able to measure one culture multiple times for example at two different time points. Another advantage is the measurement via photometer which makes the samples quantitive compareable.
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 SEAo 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. 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, therfore we dont have to destroy our cells to meaure but just take a sample from the supernatant. Also we are able to measure one culture multiple times for example 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 witten by the lab of professor Weber.
