Team:Freiburg/Project/Experiments
From 2012.igem.org
Experiments
Gene activation
![](http://imageshack.us/a/img189/6140/seapplasmid.png)
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 cell culture 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].
![]() |
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 (the latter 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 efficiency of our GATE assembly platform
Varying Cycle number of GATE assembly has limited effect
Golden Gate protocols published so far for multistep TALE assembly differ significantly in the number of digestion-ligation cycles performed. To asses, whether the cycle number significantly affects outcome, we performed 4 different GATE assemblies, each with three different cycle numbers (50, 25 and 13 cycles). We did not see significant differences in the number of colonies for the three cycle numbers. Consequently, from that point on, we performed GATE assembly with only 13 cycles (which only take approximately 2.5 hours instead of 8.5 hours for 50 cycles).
![]() |
Direpeat Amplification by Colony PCR
![Direpeat amplification by colony PCR](https://static.igem.org/mediawiki/2012/0/04/Direpeat_amp.jpg)
Activation of transcription
![SEAP essay using the TAL transcription factor plasmid targeting a minimal promotor coupled to a SEAP reporter gene](https://static.igem.org/mediawiki/2012/8/84/IGEMres4.png)
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. 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. The yellow highlighted fields show 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 statistically significant manner.
![SEAP assay using the TAL transcription factor plasmid targeting a minimal promotor coupled to a SEAP reporter gene](https://static.igem.org/mediawiki/2012/a/a6/Igemres-p.png)
After addition of pNPP, the substrate of SEAP, the activity of SEAP was measured over a period of 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:
![SEAP assay using the TAL transcription factor plasmid targeting a minimal promotor coupled to a SEAP reporter gene](https://static.igem.org/mediawiki/2012/5/55/TALTF-SEAP-TIME.png)
Reference
1. Briggs, A. W. et al. Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers. Nucl Acids Res (2012).doi:10.1093/nar/gks624