http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=20&target=Davsie&year=&month=2012.igem.org - User contributions [en]2024-03-28T21:34:03ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Freiburg/Project/ExperimentsTeam:Freiburg/Project/Experiments2012-10-26T23:09:50Z<p>Davsie: </p>
<hr />
<div>{{Template:Team:Freiburg}}<br />
__NOTOC__<br />
= Experiments =<br />
----<br />
<br />
<br />
== Gene activation ==<br />
----<br />
<html><br />
<p><br><br />
<br />
<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 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 absorps light at 405 nm and can be measured via photometry. <br />
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. <img src="http://imageshack.us/a/img189/6140/seapplasmid.png" align="right" padding:0px width="450px" hspace="20"/> 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.<br />
<br><br><br><br />
</html><br />
<br />
== Experimental design ==<br />
----<br />
<html><br />
<p><br><br />
<img src="http://imageshack.us/a/img268/53/exp1design2.png" align="left" padding:0px width="250px" hspace="20" /><br />
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. <br />
<br><br><br><br><br><br><br><br><br />
</html><br />
<br />
= Results =<br />
----<br />
<br><div align="justify">The result of our lab work was mainly the GATE assembly toolkit and the corresponding vectors. Further experiments were performed to validate the function of the kit both ''in vitro'' and ''in vivo''. <br />
<br />
<br />
== The Toolkit ==<br />
----<br />
<br><br />
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 [[Team:Freiburg/Parts|parts section]] or go to the [http://partsregistry.org Registry of Standard Biological Parts].<br />
<br />
<br />
{|align="center"<br />
|[[Image:AA1sequence.png|400px|no frame|link=]]<br />
|}<br />
<br />
<br />
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.<br />
<br><br><br />
<br />
== Creation of TAL sequences - Golden Gate Cloning ==<br />
----<br />
<br><br />
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<br />
<br />
<br><br />
== Varying Cycle number of GATE assembly has limited effect ==<br />
----<br />
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).<br><br><br />
{|align="center"<br />
|[[Image:colonies.png|400px|no frame|link=]]<br />
|}<br />
<br><br><br />
<br />
== Direpeat Amplification by Colony PCR ==<br />
----<br />
<br><br />
<html><br />
<div align="justify">To assess if the direpeats have indeed been successfully cloned into our expression vector, we have accomplished colony PCR with a variety of samples. To this end, we have designed primers which bind to both ends of the direpeat region and thus amplify the direpeats of our TAL protein. <br />
The original vector contains a kill cassette which kills bacteria unless it is replaced by the direpeats during the Golden Gate Cloning reaction. <br />
This cassette will also be amplified by the designed primers. A distinction between the amplification of the kill cassette and that of direpeats can be made upon the amplicon length: if the kill cassette is amplified, the resulting amplicon will contain 1527bp, while direpeat amplification will produce amplicons of 1276bp length. The difference between these two amplicons is 251bp, and can be detected by agarose gel electrophoresis. <br />
<br />
<img src="https://static.igem.org/mediawiki/2012/0/04/Direpeat_amp.jpg" align="right" width="400px" hspace="20" vspace="20" alt="Direpeat amplification by colony PCR"/><br />
<br />
The figure clearly demonstrates the difference in size between the amplicons of negative control (kill cassette still in the vector) and positive (direpeats have replaced the cassette) samples. While lane 2 shows a negative result with a single band of bigger size, all the other samples yielded amplicons of smaller length and thus are considered as positive due to amplification of direpeats.<br />
Nevertheless, it is obvious that the colony PCR did not produce one single product when amplifying the direpeats, but rather a smear consisting of amplicons with varying lengths. <br />
This effect is due to numerous homologies within the direpeats and has previously been described by Briggs et al.<sup>1</sup> . <br />
To eliminate those homologies to the greatest extent possible, we changed codon usage within our direpeats. Nevertheless, as the results of our colony PCR demonstrate, there is still a profound amount of homologies left, which imposes difficulties on the amplification of the direpeats array by PCR and instead results in a smear.<br />
Thus, the existence of this smear indicates the presence of direpeats within the expression vector. Moreover, the light bands at 1276 bp indicates, that the right number of direpeats have been inserted into the vector. We performed 30 colony PCRs from colonies of different GATE assemblies and had no negative results (but in some cases, we could not determine the hight of the light band). Since positive results in colony PCR of TALEs do not exclude wrong order of direpeats, we sequenced 28 clones of different GATE assemblies and analyzed the results: in 27 of the 28 clones, sequencing results entirely matched the right sequence. So the efficiency of GATE assembly is approximately 96 %.<br />
<br><br><br><br />
<br><br />
</html><br />
<br />
== Activation of transcription ==<br />
----<br />
<html><br />
<br><br />
<div align="justify">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 two to photometric analysis.<br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/8/84/IGEMres4.png" width="400px" hspace="20" vspace="20" alt="SEAP essay using the TAL transcription factor plasmid targeting a minimal promotor coupled to a SEAP reporter gene" style="margin-left:170px"/><br />
<br><br><br><br />
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.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/a/a6/Igemres-p.png" width="400px" hspace="20" vspace="20" alt="SEAP assay using the TAL transcription factor plasmid targeting a minimal promotor coupled to a SEAP reporter gene" style="margin-left:170px"/><br />
<br><br />
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).<br />
<br><br />
In the samples, that had been taken 48h after double transfection, the same effects could be demonstrated. <br />
<br><br />
Furthermore, we reapeated the same experiment for a second time. The corresponding data can be viewed here: <html><div style=text-indent:0px><a href="https://static.igem.org/mediawiki/2012/9/9c/Second_Essay.pdf">Second Experiment.</a><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/5/55/TALTF-SEAP-TIME.png" align="middle" width="500px" hspace="20" vspace="20" alt="SEAP assay using the TAL transcription factor plasmid targeting a minimal promotor coupled to a SEAP reporter gene" style="margin-left:120px"/><br />
</html><br />
<br />
== Reference ==<br />
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<br />
<br />
<br><br><br><br />
[[#top|Back to top]]</div>Davsiehttp://2012.igem.org/Team:Freiburg/Project/TalTeam:Freiburg/Project/Tal2012-10-26T21:38:51Z<p>Davsie: </p>
<hr />
<div>{{Template:Team:Freiburg}}<br />
__NOTOC__<br />
= Using the Toolkit =<br />
----<br />
<br><br />
<div align="justify">Here, we give you a manual on how to use our toolkit to design TAL proteins. We recommend reading through all of the manual prior to using the toolkit. Also we included a short introductional video on how to use the toolkit.<br />
<br />
<html><br />
<br />
<br><br><br />
<iframe style="margin-left:200px; align:center;" src="http://player.vimeo.com/video/52254697" width="400" height="300" align="middle" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe><br />
<br><br><br><br><br />
</html><br />
= Step 1. Choosing effector and target sequence =<br />
----<br />
<br>First, you need to think about your experimental setup. When working with TAL proteins it's pretty clear you want to target a DNA sequence. To choose your sequence, you need to know some of the operational details of TAL proteins in order to pick it the right way. <br />
<br />
<br />
<b>1. Every TAL binding site starts and ends with a thymine</b><br />
<br />
These thymine binding modules are already inserted in our expression plasmids. So the protein won't bind to other sequences than those which start with a T and end with a T.<br />
<br />
<br />
<b>2. Your sequence must be twelve base pair long</b><br />
<br />
Our toolbox is optimized for sequences of twelve plus two (the thymine at upstream and downstream positions). This lenght guarantees a high specifity and a library size that's good to handle at the same time.<br />
<br />
<br />
You can check out the following online softwares for perfect TAL-TF or TALEN binding sites:<br><br><br />
https://boglab.plp.iastate.edu/node/add/talen (for TALENs)<br><br />
https://boglab.plp.iastate.edu/node/add/single-tale (for TAL-TFs)<br />
<br />
<br><br />
<br />
<br><br />
<br />
= Step 2. Building a TAL =<br />
----<br />
<br>Building your TAL starts with your selected sequence. In this manual, we use a fictive sequence that you can substitute with your own. <p>Our sequence will be as follows:</p><br />
<br />
<br />
[[Image:sequence1.png|200px|center|no frame|link=]]<br />
<br />
<br />
<br />
<br>Because the two thymines are already in the cloning vector, they are of no interest for our TAL protein:<br />
<br />
[[Image:sequence2.png|200px|center|no frame|link=]]<br />
<br />
<br />
<br>To build this sequence from our toolkit we need to split it up in pairs of two:<br />
<br />
<br />
[[Image:sequence3.png|350px|center|no frame|link=]]<br />
<br />
<br>Now we need to give our pairs position numbers inside the TAL protein:<br />
<br />
<br />
[[Image:sequence4.png|500px|center|no frame|link=]]<br />
<br />
<br />
<br />
<br>Now we can start taking the parts out of the toolkit. A short look at the toolkit shows you that for every possible pair of bases, for example AA, we have 6 places. Every place stands for one of the six possible positions of the pair AA inside the TAL protein.<br />
<br />
<br />
[[Image:toolkit3.png|300px|center|no frame|link=]]<br />
<br />
<br />
<br>All you have to do now is pick the six direpeats consistent with the six pairs of your sequence. In our case, we would take the the first one of AA because the first pair of bases in our sequence is AA. Then we take the second one of TG the third of AG and so forth. The idea behind this is that every direpeat knows through his downstream and upstream part at which position of the final TAL protein it ought to be. You can find the exact mechanisms behind this in the [[Team:Freiburg/Project/Overview|'GATE Assembly Kit']] part of our project section. <br><br><br />
<br />
[[Image:sequence5.png|500px|center|no frame|link=]]<br />
<br />
<br />
<br><br />
For lazy iGEM students, we have written a simple program. So you just have to type in your target DNA sequence and we give you a list of parts that you need to pipet into your Golden Gate reaction mix:<br><br><br />
<br />
<br />
<html><br />
<div align="center"><br />
<iframe src="http://omnibus.uni-freiburg.de/~lb125/index.html"; width=80%; frameborder="0"; scrolling="no"><br />
</iframe><br />
</div><br />
</html><br />
<br />
<br />
<br />
<br><br />
<br />
= Step 3. Adding a Function =<br />
----<br />
<br><br />
Now that you have your TAL BioBricks, you are almost done. But targeting a sequence without doing anything is not really helpful, so you need a fusion protein that does something to your DNA. There are a couple of things you could do with your target sequence, and normally you have thought of this before you chose your sequence. With our toolkit you get a transcription factor to turn on or enhance the trancription of a gene and a restriction enzyme to make cuts wherever you want. Every one of these factors is already placed inside the final TAL vector and designed to fit the 3'-end of your TAL BioBricks. Conveniently, you just choose one and put it in your reaction tube along with the other BioBricks.<br />
<br />
<br />
<br />
[[Image:TALfunction.png|600px|center|no frame|link=]]<br />
<br />
<br />
<br />
With the six TAL BioBricks and the fusion enzyme in your reaction tube you now only need the type two restriction enzyme BsmB1 and a T7 Ligase to put all the parts together.<br><br><br />
<br />
[[Image:protocolggc.png|300px|left|no frame|link=]]<br />
<br />
<br><br />
<br />
[[Image:thermocycler.png|200px|center|no frame|link=]]<br />
<br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
= Step 4. Transformation and Use =<br />
----<br />
<br><br />
Transform 5 μl of the GATE assembly product into 50 μl of transformation competent bacteria.<br> <br />
<br>'''Important note:''' Your cells need to be sensitive to the ccdB kill cassette in our TAL expression vectors! Otherwise also bacteria that have taken up plasmids without the six direpeats will form false positive colonies. We used the DH10B E.coli strain.<br><br />
In case you want to express your TALE in bacteria, you need to induce the promoter of our prokaryotic expression plasmid with IPTG. <br>For use in a eukaryotic system, such as HEK 239 cells, perform a midiprep and directly transfect the eukaryotic TAL expression plasmid (or its derivatives pTAL-TF, pTALEN etc.) according to your transfection protocol. <br />
<!--- The Mission, Experiments ---><br />
<br><br><br><br><br><br><br />
[[#top|Back to top]]</div>Davsiehttp://2012.igem.org/Team:Freiburg/Project/TalTeam:Freiburg/Project/Tal2012-10-26T21:18:43Z<p>Davsie: </p>
<hr />
<div>{{Template:Team:Freiburg}}<br />
__NOTOC__<br />
= Using the Toolkit =<br />
----<br />
<br><br />
<div align="justify">Here, we give you a manual on how to use our toolkit to design TAL proteins. We recommend reading through all of the manual prior to using the toolkit. Also we included a short introductional video on how to use the toolkit.<br />
<br />
<html><br />
<br><br><br />
<iframe style="margin-right:50px" src="http://player.vimeo.com/video/52254697" width="400" height="300" align="middle" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe><br />
<br><br><br><br><br />
</html><br />
= Step 1. Choosing effector and target sequence =<br />
----<br />
<br>First, you need to think about your experimental setup. When working with TAL proteins it's pretty clear you want to target a DNA sequence. To choose your sequence, you need to know some of the operational details of TAL proteins in order to pick it the right way. <br />
<br />
<br />
<b>1. Every TAL binding site starts and ends with a thymine</b><br />
<br />
These thymine binding modules are already inserted in our expression plasmids. So the protein won't bind to other sequences than those which start with a T and end with a T.<br />
<br />
<br />
<b>2. Your sequence must be twelve base pair long</b><br />
<br />
Our toolbox is optimized for sequences of twelve plus two (the thymine at upstream and downstream positions). This lenght guarantees a high specifity and a library size that's good to handle at the same time.<br />
<br />
<br />
You can check out the following online softwares for perfect TAL-TF or TALEN binding sites:<br><br><br />
https://boglab.plp.iastate.edu/node/add/talen (for TALENs)<br><br />
https://boglab.plp.iastate.edu/node/add/single-tale (for TAL-TFs)<br />
<br />
<br><br />
<br />
<br><br />
<br />
= Step 2. Building a TAL =<br />
----<br />
<br>Building your TAL starts with your selected sequence. In this manual, we use a fictive sequence that you can substitute with your own. <p>Our sequence will be as follows:</p><br />
<br />
<br />
[[Image:sequence1.png|200px|center|no frame|link=]]<br />
<br />
<br />
<br />
<br>Because the two thymines are already in the cloning vector, they are of no interest for our TAL protein:<br />
<br />
[[Image:sequence2.png|200px|center|no frame|link=]]<br />
<br />
<br />
<br>To build this sequence from our toolkit we need to split it up in pairs of two:<br />
<br />
<br />
[[Image:sequence3.png|350px|center|no frame|link=]]<br />
<br />
<br>Now we need to give our pairs position numbers inside the TAL protein:<br />
<br />
<br />
[[Image:sequence4.png|500px|center|no frame|link=]]<br />
<br />
<br />
<br />
<br>Now we can start taking the parts out of the toolkit. A short look at the toolkit shows you that for every possible pair of bases, for example AA, we have 6 places. Every place stands for one of the six possible positions of the pair AA inside the TAL protein.<br />
<br />
<br />
[[Image:toolkit3.png|300px|center|no frame|link=]]<br />
<br />
<br />
<br>All you have to do now is pick the six direpeats consistent with the six pairs of your sequence. In our case, we would take the the first one of AA because the first pair of bases in our sequence is AA. Then we take the second one of TG the third of AG and so forth. The idea behind this is that every direpeat knows through his downstream and upstream part at which position of the final TAL protein it ought to be. You can find the exact mechanisms behind this in the [[Team:Freiburg/Project/Overview|'GATE Assembly Kit']] part of our project section. <br><br><br />
<br />
[[Image:sequence5.png|500px|center|no frame|link=]]<br />
<br />
<br />
<br><br />
For lazy iGEM students, we have written a simple program. So you just have to type in your target DNA sequence and we give you a list of parts that you need to pipet into your Golden Gate reaction mix:<br><br><br />
<br />
<br />
<html><br />
<div align="center"><br />
<iframe src="http://omnibus.uni-freiburg.de/~lb125/index.html"; width=80%; frameborder="0"; scrolling="no"><br />
</iframe><br />
</div><br />
</html><br />
<br />
<br />
<br />
<br><br />
<br />
= Step 3. Adding a Function =<br />
----<br />
<br><br />
Now that you have your TAL BioBricks, you are almost done. But targeting a sequence without doing anything is not really helpful, so you need a fusion protein that does something to your DNA. There are a couple of things you could do with your target sequence, and normally you have thought of this before you chose your sequence. With our toolkit you get a transcription factor to turn on or enhance the trancription of a gene and a restriction enzyme to make cuts wherever you want. Every one of these factors is already placed inside the final TAL vector and designed to fit the 3'-end of your TAL BioBricks. Conveniently, you just choose one and put it in your reaction tube along with the other BioBricks.<br />
<br />
<br />
<br />
[[Image:TALfunction.png|600px|center|no frame|link=]]<br />
<br />
<br />
<br />
With the six TAL BioBricks and the fusion enzyme in your reaction tube you now only need the type two restriction enzyme BsmB1 and a T7 Ligase to put all the parts together.<br><br><br />
<br />
[[Image:protocolggc.png|300px|left|no frame|link=]]<br />
<br />
<br><br />
<br />
[[Image:thermocycler.png|200px|center|no frame|link=]]<br />
<br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
= Step 4. Transformation and Use =<br />
----<br />
<br><br />
Transform 5 μl of the GATE assembly product into 50 μl of transformation competent bacteria.<br> <br />
<br>'''Important note:''' Your cells need to be sensitive to the ccdB kill cassette in our TAL expression vectors! Otherwise also bacteria that have taken up plasmids without the six direpeats will form false positive colonies. We used the DH10B E.coli strain.<br><br />
In case you want to express your TALE in bacteria, you need to induce the promoter of our prokaryotic expression plasmid with IPTG. <br>For use in a eukaryotic system, such as HEK 239 cells, perform a midiprep and directly transfect the eukaryotic TAL expression plasmid (or its derivatives pTAL-TF, pTALEN etc.) according to your transfection protocol. <br />
<!--- The Mission, Experiments ---><br />
<br><br><br><br><br><br><br />
[[#top|Back to top]]</div>Davsiehttp://2012.igem.org/Team:Freiburg/Project/TalTeam:Freiburg/Project/Tal2012-10-26T20:59:41Z<p>Davsie: </p>
<hr />
<div>{{Template:Team:Freiburg}}<br />
__NOTOC__<br />
= Using the Toolkit =<br />
----<br />
<br><br />
<div align="justify">Here, we give you a manual on how to use our toolkit to design TAL proteins. We recommend reading through all of the manual prior to using the toolkit. Also we included a short introductional video on how to use the toolkit.<br />
<br />
<html><br />
<iframe marginright="250"; src="http://player.vimeo.com/video/52254697?badge=0" width="500" height="400" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></html><br />
<br />
= Step 1. Choosing effector and target sequence =<br />
----<br />
<br>First, you need to think about your experimental setup. When working with TAL proteins it's pretty clear you want to target a DNA sequence. To choose your sequence, you need to know some of the operational details of TAL proteins in order to pick it the right way. <br />
<br />
<br />
<b>1. Every TAL binding site starts and ends with a thymine</b><br />
<br />
These thymine binding modules are already inserted in our expression plasmids. So the protein won't bind to other sequences than those which start with a T and end with a T.<br />
<br />
<br />
<b>2. Your sequence must be twelve base pair long</b><br />
<br />
Our toolbox is optimized for sequences of twelve plus two (the thymine at upstream and downstream positions). This lenght guarantees a high specifity and a library size that's good to handle at the same time.<br />
<br />
<br />
You can check out the following online softwares for perfect TAL-TF or TALEN binding sites:<br><br><br />
https://boglab.plp.iastate.edu/node/add/talen (for TALENs)<br><br />
https://boglab.plp.iastate.edu/node/add/single-tale (for TAL-TFs)<br />
<br />
<br><br />
<br />
<br><br />
<br />
= Step 2. Building a TAL =<br />
----<br />
<br>Building your TAL starts with your selected sequence. In this manual, we use a fictive sequence that you can substitute with your own. <p>Our sequence will be as follows:</p><br />
<br />
<br />
[[Image:sequence1.png|200px|center|no frame|link=]]<br />
<br />
<br />
<br />
<br>Because the two thymines are already in the cloning vector, they are of no interest for our TAL protein:<br />
<br />
[[Image:sequence2.png|200px|center|no frame|link=]]<br />
<br />
<br />
<br>To build this sequence from our toolkit we need to split it up in pairs of two:<br />
<br />
<br />
[[Image:sequence3.png|350px|center|no frame|link=]]<br />
<br />
<br>Now we need to give our pairs position numbers inside the TAL protein:<br />
<br />
<br />
[[Image:sequence4.png|500px|center|no frame|link=]]<br />
<br />
<br />
<br />
<br>Now we can start taking the parts out of the toolkit. A short look at the toolkit shows you that for every possible pair of bases, for example AA, we have 6 places. Every place stands for one of the six possible positions of the pair AA inside the TAL protein.<br />
<br />
<br />
[[Image:toolkit3.png|300px|center|no frame|link=]]<br />
<br />
<br />
<br>All you have to do now is pick the six direpeats consistent with the six pairs of your sequence. In our case, we would take the the first one of AA because the first pair of bases in our sequence is AA. Then we take the second one of TG the third of AG and so forth. The idea behind this is that every direpeat knows through his downstream and upstream part at which position of the final TAL protein it ought to be. You can find the exact mechanisms behind this in the [[Team:Freiburg/Project/Overview|'GATE Assembly Kit']] part of our project section. <br><br><br />
<br />
[[Image:sequence5.png|500px|center|no frame|link=]]<br />
<br />
<br />
<br><br />
For lazy iGEM students, we have written a simple program. So you just have to type in your target DNA sequence and we give you a list of parts that you need to pipet into your Golden Gate reaction mix:<br><br><br />
<br />
<br />
<html><br />
<div align="center"><br />
<iframe src="http://omnibus.uni-freiburg.de/~lb125/index.html"; width=80%; frameborder="0"; scrolling="no"><br />
</iframe><br />
</div><br />
</html><br />
<br />
<br />
<br />
<br><br />
<br />
= Step 3. Adding a Function =<br />
----<br />
<br><br />
Now that you have your TAL BioBricks, you are almost done. But targeting a sequence without doing anything is not really helpful, so you need a fusion protein that does something to your DNA. There are a couple of things you could do with your target sequence, and normally you have thought of this before you chose your sequence. With our toolkit you get a transcription factor to turn on or enhance the trancription of a gene and a restriction enzyme to make cuts wherever you want. Every one of these factors is already placed inside the final TAL vector and designed to fit the 3'-end of your TAL BioBricks. Conveniently, you just choose one and put it in your reaction tube along with the other BioBricks.<br />
<br />
<br />
<br />
[[Image:TALfunction.png|600px|center|no frame|link=]]<br />
<br />
<br />
<br />
With the six TAL BioBricks and the fusion enzyme in your reaction tube you now only need the type two restriction enzyme BsmB1 and a T7 Ligase to put all the parts together.<br><br><br />
<br />
[[Image:protocolggc.png|300px|left|no frame|link=]]<br />
<br />
<br><br />
<br />
[[Image:thermocycler.png|200px|center|no frame|link=]]<br />
<br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
= Step 4. Transformation and Use =<br />
----<br />
<br><br />
Transform 5 μl of the GATE assembly product into 50 μl of transformation competent bacteria.<br> <br />
<br>'''Important note:''' Your cells need to be sensitive to the ccdB kill cassette in our TAL expression vectors! Otherwise also bacteria that have taken up plasmids without the six direpeats will form false positive colonies. We used the DH10B E.coli strain.<br><br />
In case you want to express your TALE in bacteria, you need to induce the promoter of our prokaryotic expression plasmid with IPTG. <br>For use in a eukaryotic system, such as HEK 239 cells, perform a midiprep and directly transfect the eukaryotic TAL expression plasmid (or its derivatives pTAL-TF, pTALEN etc.) according to your transfection protocol. <br />
<!--- The Mission, Experiments ---><br />
<br><br><br><br><br><br><br />
[[#top|Back to top]]</div>Davsiehttp://2012.igem.org/Team:Freiburg/Project/TalTeam:Freiburg/Project/Tal2012-10-26T20:58:31Z<p>Davsie: </p>
<hr />
<div>{{Template:Team:Freiburg}}<br />
__NOTOC__<br />
= Using the Toolkit =<br />
----<br />
<br><br />
<div align="justify">Here, we give you a manual on how to use our toolkit to design TAL proteins. We recommend reading through all of the manual prior to using the toolkit. Also we included a short introductional video on how to use the toolkit.<br />
<br />
<html><br />
<iframe src="http://player.vimeo.com/video/52254697?badge=0" width="500" height="400" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe> <p><a href="http://vimeo.com/52254697">How to use the GATE-kit</a> from <a href="http://vimeo.com/user13636063">iGem Freiburg</a> on <a href="http://vimeo.com">Vimeo</a>.</p></html><br />
<br />
= Step 1. Choosing effector and target sequence =<br />
----<br />
<br>First, you need to think about your experimental setup. When working with TAL proteins it's pretty clear you want to target a DNA sequence. To choose your sequence, you need to know some of the operational details of TAL proteins in order to pick it the right way. <br />
<br />
<br />
<b>1. Every TAL binding site starts and ends with a thymine</b><br />
<br />
These thymine binding modules are already inserted in our expression plasmids. So the protein won't bind to other sequences than those which start with a T and end with a T.<br />
<br />
<br />
<b>2. Your sequence must be twelve base pair long</b><br />
<br />
Our toolbox is optimized for sequences of twelve plus two (the thymine at upstream and downstream positions). This lenght guarantees a high specifity and a library size that's good to handle at the same time.<br />
<br />
<br />
You can check out the following online softwares for perfect TAL-TF or TALEN binding sites:<br><br><br />
https://boglab.plp.iastate.edu/node/add/talen (for TALENs)<br><br />
https://boglab.plp.iastate.edu/node/add/single-tale (for TAL-TFs)<br />
<br />
<br><br />
<br />
<br><br />
<br />
= Step 2. Building a TAL =<br />
----<br />
<br>Building your TAL starts with your selected sequence. In this manual, we use a fictive sequence that you can substitute with your own. <p>Our sequence will be as follows:</p><br />
<br />
<br />
[[Image:sequence1.png|200px|center|no frame|link=]]<br />
<br />
<br />
<br />
<br>Because the two thymines are already in the cloning vector, they are of no interest for our TAL protein:<br />
<br />
[[Image:sequence2.png|200px|center|no frame|link=]]<br />
<br />
<br />
<br>To build this sequence from our toolkit we need to split it up in pairs of two:<br />
<br />
<br />
[[Image:sequence3.png|350px|center|no frame|link=]]<br />
<br />
<br>Now we need to give our pairs position numbers inside the TAL protein:<br />
<br />
<br />
[[Image:sequence4.png|500px|center|no frame|link=]]<br />
<br />
<br />
<br />
<br>Now we can start taking the parts out of the toolkit. A short look at the toolkit shows you that for every possible pair of bases, for example AA, we have 6 places. Every place stands for one of the six possible positions of the pair AA inside the TAL protein.<br />
<br />
<br />
[[Image:toolkit3.png|300px|center|no frame|link=]]<br />
<br />
<br />
<br>All you have to do now is pick the six direpeats consistent with the six pairs of your sequence. In our case, we would take the the first one of AA because the first pair of bases in our sequence is AA. Then we take the second one of TG the third of AG and so forth. The idea behind this is that every direpeat knows through his downstream and upstream part at which position of the final TAL protein it ought to be. You can find the exact mechanisms behind this in the [[Team:Freiburg/Project/Overview|'GATE Assembly Kit']] part of our project section. <br><br><br />
<br />
[[Image:sequence5.png|500px|center|no frame|link=]]<br />
<br />
<br />
<br><br />
For lazy iGEM students, we have written a simple program. So you just have to type in your target DNA sequence and we give you a list of parts that you need to pipet into your Golden Gate reaction mix:<br><br><br />
<br />
<br />
<html><br />
<div align="center"><br />
<iframe src="http://omnibus.uni-freiburg.de/~lb125/index.html"; width=80%; frameborder="0"; scrolling="no"><br />
</iframe><br />
</div><br />
</html><br />
<br />
<br />
<br />
<br><br />
<br />
= Step 3. Adding a Function =<br />
----<br />
<br><br />
Now that you have your TAL BioBricks, you are almost done. But targeting a sequence without doing anything is not really helpful, so you need a fusion protein that does something to your DNA. There are a couple of things you could do with your target sequence, and normally you have thought of this before you chose your sequence. With our toolkit you get a transcription factor to turn on or enhance the trancription of a gene and a restriction enzyme to make cuts wherever you want. Every one of these factors is already placed inside the final TAL vector and designed to fit the 3'-end of your TAL BioBricks. Conveniently, you just choose one and put it in your reaction tube along with the other BioBricks.<br />
<br />
<br />
<br />
[[Image:TALfunction.png|600px|center|no frame|link=]]<br />
<br />
<br />
<br />
With the six TAL BioBricks and the fusion enzyme in your reaction tube you now only need the type two restriction enzyme BsmB1 and a T7 Ligase to put all the parts together.<br><br><br />
<br />
[[Image:protocolggc.png|300px|left|no frame|link=]]<br />
<br />
<br><br />
<br />
[[Image:thermocycler.png|200px|center|no frame|link=]]<br />
<br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
= Step 4. Transformation and Use =<br />
----<br />
<br><br />
Transform 5 μl of the GATE assembly product into 50 μl of transformation competent bacteria.<br> <br />
<br>'''Important note:''' Your cells need to be sensitive to the ccdB kill cassette in our TAL expression vectors! Otherwise also bacteria that have taken up plasmids without the six direpeats will form false positive colonies. We used the DH10B E.coli strain.<br><br />
In case you want to express your TALE in bacteria, you need to induce the promoter of our prokaryotic expression plasmid with IPTG. <br>For use in a eukaryotic system, such as HEK 239 cells, perform a midiprep and directly transfect the eukaryotic TAL expression plasmid (or its derivatives pTAL-TF, pTALEN etc.) according to your transfection protocol. <br />
<!--- The Mission, Experiments ---><br />
<br><br><br><br><br><br><br />
[[#top|Back to top]]</div>Davsiehttp://2012.igem.org/Team:FreiburgTeam:Freiburg2012-10-26T20:54:39Z<p>Davsie: </p>
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<h1>Let us tell you a fabulous TALE...</h1><br />
Transactivator-Like Effectors (TALEs) are a brand-new technology that currently revolutionizes the way researchers manipulate DNA with exceptional site specificity. Originally derived from ''Xanthomonas spp.'', this type of protein comprizes an effector domain and a modular DNA binding domain that can be rationally designed to bind to virtually any target sequence of DNA. Over the past two years, universal endonucleases (TALENs) and transcription factors have been tested in various organisms ranging from bacteria to humans. According to existing protocols, TALE assembly requires several weeks of work and substantial lab skills. In order to bring this technology within reach for iGEM students, we invented an extremely fast and easy TALE assembly strategy and developed a TALE platform with expression plasmids and new classes of TALEs. With our so called GATE assembly kit, future iGEM students will be able to precisely manipulate genomic loci easier and faster than anyone else in the world.<br />
</div><br />
<br />
<div class="slide"><br />
<h1>European iGEM Jamboree 2012 Amsterdam:</h1><br />
Team freiGEM won a gold medal and a special prize for the 'Best New BioBrick Part or Device, Engineered'! Team freiGEM also advanced to World Championship in Boston!<br />
<p align="center"><A HREF="http://www.flickr.com/photos/igemeurope/8096765971/in/photostream/" target="_blank"><img class="thumbnail" img src='http://omnibus.uni-freiburg.de/~lb125/won1.png' width="75%" height="75%"/></A></p><br />
</div><br />
<br />
<div class="slide"><br />
<h1><iframe align="right" width="300" height="250" vspace="10" hspace="20" src="http://player.vimeo.com/video/49902809" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe>Watch our freiGEM movie...<h1><br />
<br><br />
<h1>...or get some impressions in the freiGEM Photo Gallery:</h1><br />
<p><a style="font-weight:bold; font-size:1.3em;" href="/Team:Freiburg/gallery">FreiGEM 2012 Gallery</a></p><br />
</div><br />
<br />
<div class="slide"><br />
<h1><iframe align="left" width="300" height="250" <iframe src="http://player.vimeo.com/video/52254697?badge=0" width="500" height="400" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe>See how easy it is to get your own custom made TAL...</h1><br />
<br><br />
<h1>For further information visit our Project page:</h1><br />
<p><a style="font-weight:bold; font-size:1.3em;" href="/Team:Freiburg/Project">Overview TAL Project</a></p><br />
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With kind support of<br />
<br><br />
<br><br />
</span><br />
<A HREF="http://www.bioss.uni-freiburg.de/cms/index.php" target="_blank"><img class="thumbnail" img src="https://static.igem.org/mediawiki/2012/7/7b/Logo_bioss.gif" width= "200" /></A><br />
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<img class="thumbnail" img src='http://omnibus.uni-freiburg.de/~lb125/metabion.png' width= "180"/><br />
<A HREF="http://www.genscript.com/" target="_blank"><img class="thumbnail" img src='https://static.igem.org/mediawiki/2012/0/0d/Genscript_logo.gif' width= "180"/></A><br />
<A HREF="http://eu.idtdna.com/site" target="_blank"><img class="thumbnail" img src='https://static.igem.org/mediawiki/2012/2/21/IDTLogo2010.png' width= "200"/></A><br />
<A HREF="http://www.lifetechnologies.com" target="_blank"><img class="thumbnail" img src='http://omnibus.uni-freiburg.de/~lb125/life.png' width= "150"/></A><br />
<A HREF="http://www.eurofinsdna.com/home.html" target="_blank"><img class="thumbnail" img src='http://omnibus.uni-freiburg.de/~lb125/eurofins.jpg' width= "150"/></A><br />
<img class="thumbnail" img src='http://omnibus.uni-freiburg.de/~lb125/gatc.png' width= "150"/><br />
<A HREF="http://www.erasynbio.net/" target="_blank"><img class="thumbnail" img src='http://omnibus.uni-freiburg.de/~lb125/erasynbio.png' width= "200"/></A><br />
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<br />
<!--- The Mission, Experiments ---></div>Davsiehttp://2012.igem.org/Team:Freiburg/Project/IntroTeam:Freiburg/Project/Intro2012-10-26T20:05:29Z<p>Davsie: </p>
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__NOTOC__<br />
= Introduction =<br />
----<br />
<html><br />
<!-- BEGIN TIMELINE WHITEBOX HERE --><br />
<div name="talhistory"><br />
<div id="timeline"><br />
<br />
<ul id="dates"><br />
<li><a href="#" class="dateobject">2009</a></li><br />
<li><a href="#" class="dateobject">2010</a></li><br />
<li><a href="#" class="dateobject">02/11</a></li><br />
<li><a href="#" class="dateobject">10/11</a></li><br />
<li><a href="#" class="dateobject">12/11</a></li><br />
<li><a href="#" class="dateobject">02/12</a></li><br />
<li><a href="#" class="dateobject">04/12</a></li><br />
<li><a href="#" class="dateobject">iGEM'12</a></li><br />
</ul><br />
<br />
<br />
<div id="grad_left"></div><br />
<br />
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<br />
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<br />
<br />
<br />
<li id="#2009"><br />
<br />
<img src="http://omnibus.uni-freiburg.de/~lb125/10_09.png" width="256" height="170" /><br />
<br />
<div class="issuedate" style="font-weight:bold; font-size:1.3em;">October 2009</div><br />
<br />
<p>Two research groups publish the TAL Effector codes in the same issue of Science: Amino acid 12 and 13 of every DNA binding module specifically binds to one nucleotide</p><br />
<br />
</li><br />
<br />
<br />
<li id="#2010"><br />
<br />
<img src="http://omnibus.uni-freiburg.de/~lb125/10_10.png" width="256" height="200" /><br />
<br />
<div class="issuedate" style="font-weight:bold; font-size:1.3em;">October 2010</div><br />
<br />
<p>Voytas Lab develops TALENs. These fusion proteins of FokI and a TAL protein cut as dimers and allow researchers to cut virtually anywhere in the genome. Since double strand breaks increase efficiency of homologous recombination, TALENS are a powerful tool for genetic engineering and gene therapy</p><br />
<br />
</li><br />
<br />
<li id="#02/2011"><br />
<br />
<img src="http://omnibus.uni-freiburg.de/~lb125/02_11.png" width="256" height="256" /><br />
<br />
<div class="issuedate" style="font-weight:bold; font-size:1.3em;">February 2011</div><br />
<br />
<p>Based on an exclusive licensing agreement with the University of Minnasota, Cellectis bioresearch launches its TAL effector product line. One TALEN pair currently costs 5000 Euro (6454 US$, 26.10.12).</p><br />
<br />
</li><br />
<br />
<br />
<li id="#10/2011"><br />
<br />
<img src="http://omnibus.uni-freiburg.de/~lb125/10_11.png" width="210" height="256" /><br />
<br />
<div class="issuedate" style="font-weight:bold; font-size:1.3em;">October 2011</div><br />
<br />
<p>The iGEM team from Harvard University employed fancy and expensive techniques to find up to 15 new zinc fingers (each of which binds to 3 bp). There has to be a better way…</p><br />
<br />
</li><br />
<br />
<br />
<li id="#12/2011"><br />
<br />
<img src="http://omnibus.uni-freiburg.de/~lb125/12_11.png" width="210" height="256" /><br />
<br />
<div class="issuedate" style="font-weight:bold; font-size:1.3em;">December 2011</div><br />
<br />
<p>Nature chooses TALENs as the 2011 Method of the year.</p><br />
<br />
</li><br />
<br />
<li id="#02/2012"><br />
<br />
<img src="http://omnibus.uni-freiburg.de/~lb125/02_12.png" width="256" height="256" /><br />
<br />
<div class="issuedate" style="font-weight:bold; font-size:1.3em;">February 2012</div><br />
<br />
<p>The first two crystal structures of TALE modules bound to DNA published in the same issue of Science. The protein literally wraps itself around the DNA double helix and forms these beautiful symmetric shapes.</p><br />
<br />
</li><br />
<br />
<br />
<br />
<li id="#04/2012"><br />
<br />
<img src="http://omnibus.uni-freiburg.de/~lb125/04_12.png" width="256" height="256" /><br />
<br />
<div class="issuedate" style="font-weight:bold; font-size:1.3em;">April 2012</div><br />
<br />
<p>Joung lab publishes FLASH assembly in Nature Biotechnology. This first automatable TAL assembly platform facilitates assembly of 96 TAL DNA fragments in less than a day using a pipeting robot.</p><br />
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<div class="issuedate" style="font-weight:bold; font-size:1.3em;">October 2012</div><br />
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<p>The Freiburg iGEM team makes TALE technology available to everyone by introducing the GATE assembly kit. For TALEs targeting 14 bp, this platform is currently the fastest, cheapest and easiest method in the world.</p><br />
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<p><div align="justify">Originally, TAL proteins are virulence factors of the plant-pathogenic ''Xanthomonas spp.'' that are injected into plant cells via a type III secretion system in order to modulate transcription<sup>1</sup>. For this purpose, their c-terminal end contains a nuclear localisation signal (NLS) and an acidic activation domain. The central part of the TAL protein contains a number of almost similar repeats that mediate specific binding to target loci in the genome.<br />
In 2009, two groups have simultaneously pointed out that each of these repeats specifically binds to one base of the target DNA via two amino acids (aa 12 and 13), named the repeat variable diresidues (RVD)<sup>2</sup>. Moreover, it has been shown that DNA binding of these proteins is highly modular, i.e. the number of bases or sequence of the target DNA can be altered by adjusting the number or order of the repeats in the TAL protein, respectively.<br />
The minimal condition for TALE activity is a thymine at the 5’ end of the target sequence. Further target sequence requirements that allow for one TALEN pair binding site every 35 bp (published by the Voytas lab in 2011<sup>3</sup>) have recently been questioned by Reyon et al.<sup>4</sup> In summary, it is very likely that you can find multiple potential TALE binding sites in any sequence you want to target.<br />
This, obviously, is very promising for biotechnological and clinical applications. Thus, two major classes of TAL Effectors have been created by replacing the natural acidic activation domain either by other transcription factors (TALE-TFs) <sup>5</sup> or by a monomer of the non-sequence specific nuclease FokI, resulting in TAL Effector Nucleases (TALENS).<sup>6</sup> A pair of TALENs can be designed to bind adjacent DNA sequences in a way that the two monomers are able to form a functional FokI dimer that produces a double strand break (DBS) within the spacer between the TAL-Effectors (see figure 13). <br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/97/TAL-figure13.png" width="500px" style="margin-left:150px"/><br><div align="center">Figure 2: BsmB1 restriction mechanism</div><br><br><br />
<br />
Subsequently, the cell repairs the DBS by either non-homologous end joining (NHEJ, which results in indels at the DSB-site) or homologous recombination of exogenously added genetic material. That way, TALENs allow researchers to introduce genes into a genome with much higher efficiency than before.<br />
In this context, TALENs and TALE-TF have successfully been applied for manipulation of a series of genes in different organisms such as yeast<sup>7</sup>, tobacco<sup>3</sup>, fruitflies<sup>6,8</sup>,worms<sup>6,9</sup>, zebrafish<sup>10</sup>, rats<sup>11</sup> and various human cell types, including human stem cells<sup>12</sup>. Moreover, TALENs are already applied for gene therapy in preclinical trials.<br><br><br />
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<div style="color: #1C649F; font-size: 20px;font-family: Gill Sans MT">References</div><br><br />
1. Scholze, H. & Boch, J. TAL effectors are remote controls for gene activation. ''Current Opinion in Microbiology'' 14, 47–53 (2011).<br><br />
2. Moscou, M. J. & Bogdanove, A. J. A Simple Cipher Governs DNA Recognition by TAL Effectors. ''Science'' 326, 1501–1501 (2009).<br><br />
3. Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. ''Nucleic Acids Res'' 39, e82 (2011).<br><br />
4. Reyon, D. et al. FLASH assembly of TALENs for high-throughput genome editing. ''Nature Biotechnology'' 30, 460–465 (2012).<br><br />
5. Zhang, F. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. ''Nature biotechnology'' 29, 149–153 (2011).<br><br />
6. Miller, J. C. et al. A TALE nuclease architecture for efficient genome editing. ''Nature Biotechnology'' 29, 143–148 (2010).<br><br />
7. Boch, J. et al. Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors. ''Science'' 326, 1509–1512 (2009).<br />
8. Liu, J. et al. Efficient and Specific Modifications of the Drosophila Genome by Means of an Easy TALEN Strategy. ''Journal of Genetics and Genomics'' 39, 209–215 (2012).<br><br />
9. Wood, A. J. et al. Targeted Genome Editing Across Species Using ZFNs and TALENs. ''Science'' 333, 307–307 (2011).<br><br />
10. Sander, J. D. et al. Targeted gene disruption in somatic zebrafish cells using engineered TALENs. ''Nat Biotechnol'' 29, 697–698 (2011).<br><br />
11. Tesson, L. et al. Knockout rats generated by embryo microinjection of TALENs. ''Nature Biotechnology'' 29, 695–696 (2011).<br><br />
12. Hockemeyer, D. et al. Genetic engineering of human pluripotent cells using TALE nucleases. ''Nature Biotechnology'' 29, 731–734 (2011)<br />
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[[#top|Back to top]]</div>Davsiehttp://2012.igem.org/File:TAL-figure13.pngFile:TAL-figure13.png2012-10-26T20:00:46Z<p>Davsie: </p>
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<div></div>Davsiehttp://2012.igem.org/Team:Freiburg/Project/TalTeam:Freiburg/Project/Tal2012-10-26T19:45:56Z<p>Davsie: </p>
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<div>{{Template:Team:Freiburg}}<br />
__NOTOC__<br />
= Using the Toolkit =<br />
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<div align="justify">Here, we give you a manual on how to use our toolkit to design TAL proteins. We recommend reading through all of the manual prior to using the toolkit. Also we included a short introductional video on how to use the toolkit.<br />
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= Step 1. Choosing effector and target sequence =<br />
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<br>First, you need to think about your experimental setup. When working with TAL proteins it's pretty clear you want to target a DNA sequence. To choose your sequence, you need to know some of the operational details of TAL proteins in order to pick it the right way. <br />
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<b>1. Every TAL binding site starts and ends with a thymine</b><br />
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These thymine binding modules are already inserted in our expression plasmids. So the protein won't bind to other sequences than those which start with a T and end with a T.<br />
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<b>2. Your sequence must be twelve base pair long</b><br />
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Our toolbox is optimized for sequences of twelve plus two (the thymine at upstream and downstream positions). This lenght guarantees a high specifity and a library size that's good to handle at the same time.<br />
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You can check out the following online softwares for perfect TAL-TF or TALEN binding sites:<br><br><br />
https://boglab.plp.iastate.edu/node/add/talen (for TALENs)<br><br />
https://boglab.plp.iastate.edu/node/add/single-tale (for TAL-TFs)<br />
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= Step 2. Building a TAL =<br />
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<br>Building your TAL starts with your selected sequence. In this manual, we use a fictive sequence that you can substitute with your own. <p>Our sequence will be as follows:</p><br />
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[[Image:sequence1.png|200px|center|no frame|link=]]<br />
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<br>Because the two thymines are already in the cloning vector, they are of no interest for our TAL protein:<br />
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[[Image:sequence2.png|200px|center|no frame|link=]]<br />
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<br>To build this sequence from our toolkit we need to split it up in pairs of two:<br />
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[[Image:sequence3.png|350px|center|no frame|link=]]<br />
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<br>Now we need to give our pairs position numbers inside the TAL protein:<br />
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[[Image:sequence4.png|500px|center|no frame|link=]]<br />
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<br>Now we can start taking the parts out of the toolkit. A short look at the toolkit shows you that for every possible pair of bases, for example AA, we have 6 places. Every place stands for one of the six possible positions of the pair AA inside the TAL protein.<br />
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[[Image:toolkit3.png|300px|center|no frame|link=]]<br />
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<br>All you have to do now is pick the six direpeats consistent with the six pairs of your sequence. In our case, we would take the the first one of AA because the first pair of bases in our sequence is AA. Then we take the second one of TG the third of AG and so forth. The idea behind this is that every direpeat knows through his downstream and upstream part at which position of the final TAL protein it ought to be. You can find the exact mechanisms behind this in the [[Team:Freiburg/Project/Overview|'GATE Assembly Kit']] part of our project section. <br><br><br />
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[[Image:sequence5.png|500px|center|no frame|link=]]<br />
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For lazy iGEM students, we have written a simple program. So you just have to type in your target DNA sequence and we give you a list of parts that you need to pipet into your Golden Gate reaction mix:<br><br><br />
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= Step 3. Adding a Function =<br />
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Now that you have your TAL BioBricks, you are almost done. But targeting a sequence without doing anything is not really helpful, so you need a fusion protein that does something to your DNA. There are a couple of things you could do with your target sequence, and normally you have thought of this before you chose your sequence. With our toolkit you get a transcription factor to turn on or enhance the trancription of a gene and a restriction enzyme to make cuts wherever you want. Every one of these factors is already placed inside the final TAL vector and designed to fit the 3'-end of your TAL BioBricks. Conveniently, you just choose one and put it in your reaction tube along with the other BioBricks.<br />
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[[Image:TALfunction.png|600px|center|no frame|link=]]<br />
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With the six TAL BioBricks and the fusion enzyme in your reaction tube you now only need the type two restriction enzyme BsmB1 and a T7 Ligase to put all the parts together.<br><br><br />
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[[Image:protocolggc.png|300px|left|no frame|link=]]<br />
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[[Image:thermocycler.png|200px|center|no frame|link=]]<br />
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= Step 4. Transformation and Use =<br />
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Transform 5 μl of the GATE assembly product into 50 μl of transformation competent bacteria.<br> <br />
<br>'''Important note:''' Your cells need to be sensitive to the ccdB kill cassette in our TAL expression vectors! Otherwise also bacteria that have taken up plasmids without the six direpeats will form false positive colonies. We used the DH10B E.coli strain.<br><br />
In case you want to express your TALE in bacteria, you need to induce the promoter of our prokaryotic expression plasmid with IPTG. <br>For use in a eukaryotic system, such as HEK 239 cells, perform a midiprep and directly transfect the eukaryotic TAL expression plasmid (or its derivatives pTAL-TF, pTALEN etc.) according to your transfection protocol. <br />
<!--- The Mission, Experiments ---><br />
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[[#top|Back to top]]</div>Davsiehttp://2012.igem.org/Team:Freiburg/ProjectTeam:Freiburg/Project2012-10-26T19:42:29Z<p>Davsie: </p>
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<div>{{Template:Team:Freiburg}}<br />
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__NOTOC__<br />
= Project =<br />
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<br><br />
[[File:projectsymbolT.png|center|180px|link=]]<br />
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== Overview ==<br />
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<div align="justify">TALE technology currently revolutionizes synthetic biology, not only because of higher sequence fidelity or less cytotoxicity compared to other DNA binding proteins (e.g. zinc fingers). The main advantage is that they can be produced rationally to bind a DNA sequence of choice, whereas zinc fingers with the desired binding properties need to be selected from a library of fingers. That is why this technology is generally much less costly, time consuming and does guarantee binding sites for every predefined sequence than the zinc finger technology, although open source platforms have also been published for the latter<sup>1</sup>. <br />
Consequently, deciphering the TAL code also resulted in an enormous step towards democratizing targeted DNA manipulation<sup>2</sup>. Moreover, multiple protocols and open source kits have been published by the most influential labs in the field over the past year, which further popularized TALEs<sup>3,4,5</sup>.<br />
However, we believe that the last step of democratizing precise gene targeting has not been made yet – this hypothesis is corroborated by the fact that the biotech companies Cellectis bioresearch and Invitrogen have launched quite expensive new TAL effector product lines during the last few months.<br />
In order to bring TAL technology within reach for everyone, in particular for future iGEM students, we identified the two main bottlenecks of conventional TALE assembly, namely that it is very time consuming and requires substantial training in molecular biology.<br />
In the next steps, we invented a method, that we refer to as Golden Gate cloning- based, automatable TAL Effector (GATE) assembly, and built the genetic parts (the GATE assembly toolkit) to actually assemble custom TALEs at record speed. Furthermore, we quantified the efficiency of our GATE assembly and tested our constructs in a Human Embryonic Kidney (HEK) cell line. We are proud to say that with our GATE assembly kit, future iGEM students will be able to easily assemble custom 12.5 repeat TALEs faster than anyone else in the world.<br />
While working on the GATE assembly kit, we learned a lot about Golden Gate cloning and came up with a strategy to introduce this powerful cloning technology to the iGEM registry as the Golden Gate standard without compromising existing standards. <br />
Our major goal was to empower future iGEM students to use and further develop TALE technology. That is why we dedicated a whole subsection of our project description to a step-by-step GATE assembly protocol.<br />
We believe that by enabling virtually anyone to specifically manipulate any locus even in the context of a whole genome, we have done the last step towards democratizing gene targeting. Although to date, the GATE assembly kit is complete for only a few weeks, we regularly receive requests from research groups all over Europe, asking for copies of the kit. Moreover, we got approached by the open source plasmid repository [http://www.addgene.org/ Addgene] that wants to distribute our toolkit. We are currently preparing to send our kit to them so the GATE kit will be available to everyone soon! That way, have a significant impact also on the research world around iGEM.<br />
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We believe that we have laid a solid foundation for super-easy site specific genome modifications for future iGEM teams.<br />
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== [[Team:Freiburg/Project/Intro|Introduction]]==<br />
<div style="font-size: 12px"><br />
You don't know what TAL effectors actually are? We reviewed the recent literature for you, to give you a quick overview of this exciting new field of research.</div><br />
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== [[Team:Freiburg/Project/Golden|Golden Gate Standard]]==<br />
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<div style=" font-size: 12px;align=justify"> Assembling multiple gene constructs in frame without leaving scars is not possible with existing iGEM standards. We therefore introduce the new Golden-Gate Standard that is fully compatible with RFC 10.</div><br />
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==[[Team:Freiburg/Project/Vektor|The TAL Vector]]==<br />
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<div style=" font-size: 12px;align=justify">Targeting a sequence and not doing something to it, is like throwing mechanics at your car. Your car will not get any better only the mechanics will get mad. Because we know this, we bring the tools you need to actually work with DNA.To make it even more easy these tools are deliverd already inside the final TAL backbone, just add the sequence and you're ready.</div><br />
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== [[Team:Freiburg/Project/Overview|GATE Assembly Kit]]==<br />
<br />
<div style=" font-size: 12px;align=justify"> We have invented a super-fast, super-easy and super-cheap Method for custom TAL effector construction. Learn about the theory behind the TAL effector toolkit, how we created it and why we choose this design.</div><br />
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==[[Team:Freiburg/Project/Tal|Using the Toolkit]]==<br />
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<div style=" font-size: 12px; align=justify">Our overall goal is to empower future iGEM teams to use the most exciting new technology synthetic biology has to offer. We therefore not only invented the GATE assembly platform but wrote a step by step manual for super-easy custom TALE construction <br />
</div><br />
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==[[Team:Freiburg/Project/Robot|The Future of TAL]]==<br />
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<div style=" font-size: 12px;align=justify">Until now, almost three years after deciphering the TALE code, only two types of TAL Effectors have been developed: TALENs and TAL-TFs. We herein propose additional classes of TAL effectors.</div><br />
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==[[Team:Freiburg/Project/Experiments|Experiments and Results]]==<br />
<div style="color: #1C649F; font-size: 12px; align=justify">We not only rigorously tested if our in vitro TALE gene assembly method works but also if our TALE constructs actually work in a human cell line. Check out test design and results.</div><br />
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==References==<br />
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1. Maeder, M. L. et al. Rapid ‘Open-Source’ Engineering of Customized Zinc-Finger Nucleases for Highly Efficient Gene Modification. ''Molecular Cell'' 31, 294–301 (2008).<br><br />
2. Clark, K. J., Voytas, D. F. & Ekker, S. C. A TALE of two nucleases: gene targeting for the masses? ''Zebrafish'' 8, 147–149 (2011).<br><br />
3. Sanjana, N. E. et al. A transcription activator-like effector toolbox for genome engineering. ''Nature Protocols'' 7, 171–192 (2012).<br><br />
4. Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. ''Nucleic Acids Res'' 39, e82 (2011).<br><br />
5. Reyon, D. et al. FLASH assembly of TALENs for high-throughput genome editing. ''Nature Biotechnology'' 30, 460–465 (2012).<br><br />
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<!--- The Mission, Experiments ---></div>Davsiehttp://2012.igem.org/Team:Freiburg/Project/ExperimentsTeam:Freiburg/Project/Experiments2012-10-24T11:17:41Z<p>Davsie: </p>
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<div>{{Template:Team:Freiburg}}<br />
__NOTOC__<br />
= Experiments =<br />
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== Gene activation ==<br />
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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 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 absorps light at 405 nm and can be measured via photometry. <img src="http://imageshack.us/a/img189/6140/seapplasmid.png" align="right" padding:0px width="450px" hspace="20"/><br />
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.<br />
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== Experimental design ==<br />
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<img src="http://imageshack.us/a/img268/53/exp1design2.png" align="left" padding:0px width="250px" hspace="20" /><br />
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. <br />
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= Results =<br />
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<br><div align="justify">The result of our lab work was mainly the GATE assembly toolkit and the corresponding vectors. Further experiments were performed to validate the function of the kit both ''in vitro'' and ''in vivo''. <br />
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== The Toolkit ==<br />
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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 [[Team:Freiburg/Parts|parts section]] or go to the [http://partsregistry.org Registry of Standard Biological Parts].<br />
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{|align="center"<br />
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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.<br />
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== Creation of TAL sequences - Golden Gate Cloning ==<br />
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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<br />
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<br><br />
== Varying Cycle number of GATE assembly has limited effect ==<br />
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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).<br><br><br />
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== Direpeat Amplification by Colony PCR ==<br />
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<div align="justify">To assess if the direpeats have indeed been successfully cloned into our expression vector, we have accomplished colony PCR with a variety of samples. To this end, we have designed primers which bind to both ends of the direpeat region and thus amplify the direpeats of our TAL protein. <br />
The original vector contains a kill cassette which kills bacteria unless it is replaced by the direpeats during the Golden Gate Cloning reaction. <br />
This cassette will also be amplified by the designed primers. A distinction between the amplification of the kill cassette and that of direpeats can be made upon the amplicon length: if the kill cassette is amplified, the resulting amplicon will contain 1527bp, while direpeat amplification will produce amplicons of 1276bp length. The difference between these two amplicons is 251bp, and can be detected by agarose gel electrophoresis. (Figure)<br />
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<img src="https://static.igem.org/mediawiki/2012/0/04/Direpeat_amp.jpg" align="right" width="400px" hspace="20" vspace="20" alt="Direpeat amplification by colony PCR"/><br />
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The figure clearly demonstrates the difference in size between the amplicons of negative control (kill cassette still in the vector) and positive (direpeats have replaced the cassette) samples. While lane 2 shows a negative result with a single band of bigger size, all the other samples yielded amplicons of smaller length and thus are considered as positive due to amplification of direpeats.<br />
Nevertheless, it is obvious that the colony PCR did not produce one single product when amplifying the direpeats, but rather a smear consisting of amplicons with varying lengths. <br />
This effect is due to numerous homologies within the direpeats and has previously been described by Briggs et al.<sup>1</sup> . <br />
To eliminate those homologies to the greatest extent possible, we changed codon usage within our direpeats. Nevertheless, as the results of our colony PCR demonstrate, there is still a profound amount of homologies left, which imposes difficulties on the amplification of the direpeats array by PCR and instead results in a smear.<br />
Thus, the existence of this smear indicates the presence of direpeats within the expression vector. Moreover, the light bands at 1276 bp indicates, that the right number of direpeats have been inserted into the vector. We performed 30 colony PCRs from colonies of different GATE assemblies and had no negative results (but in some cases, we could not determine the hight of the light band). Since positive results in colony PCR of TALEs do not exclude wrong order of direpeats, we sequenced 28 clones of different GATE assemblies and analyzed the results: in 27 of the 28 clones, sequencing results entirely matched the right sequence. So the efficiency of GATE assembly is approximately 96 %.<br />
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== Activation of transcription ==<br />
----<br />
<html><br />
<br><br />
<div align="justify">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).<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/8/84/IGEMres4.png" align="right" width="400px" hspace="20" vspace="20" alt="SEAP essay using the TAL transcription factor plasmid targeting a minimal promotor coupled to a SEAP reporter gene"/><br />
<br />
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 two to photometric analysis.<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
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.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/a/a6/Igemres-p.png" width="400px" hspace="20" vspace="20" alt="SEAP assay using the TAL transcription factor plasmid targeting a minimal promotor coupled to a SEAP reporter gene" style="margin-left:170px"/><br />
<br><br />
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).<br />
<br><br />
In the samples, that had been taken 48h after double transfection, the same effects could be demonstrated. <br />
<br><br />
Furthermore, we reapeated the same experiment for a second time. The corresponding data can be viewed here: <html><div style=text-indent:0px><a href="https://static.igem.org/mediawiki/2012/9/9c/Second_Essay.pdf">Second Experiment.</a><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/5/55/TALTF-SEAP-TIME.png" align="middle" width="500px" hspace="20" vspace="20" alt="SEAP assay using the TAL transcription factor plasmid targeting a minimal promotor coupled to a SEAP reporter gene" style="margin-left:120px"/><br />
</html><br />
<br />
== Reference ==<br />
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<br />
<br />
<br><br><br><br />
[[#top|Back to top]]</div>Davsiehttp://2012.igem.org/File:Igemres-p.pngFile:Igemres-p.png2012-10-24T11:16:00Z<p>Davsie: </p>
<hr />
<div></div>Davsiehttp://2012.igem.org/Team:Freiburg/Project/ExperimentsTeam:Freiburg/Project/Experiments2012-10-23T14:18:32Z<p>Davsie: </p>
<hr />
<div>{{Template:Team:Freiburg}}<br />
__NOTOC__<br />
= Experiments =<br />
----<br />
<br />
<br />
== Gene activation ==<br />
----<br />
<html><br />
<p><br><br />
<br />
<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 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 absorps light at 405 nm and can be measured via photometry. <img src="http://imageshack.us/a/img189/6140/seapplasmid.png" align="right" padding:0px width="450px" hspace="20"/><br />
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.<br />
<br><br><br><br />
</html><br />
<br />
== Experimental design ==<br />
----<br />
<html><br />
<p><br><br />
<img src="http://imageshack.us/a/img268/53/exp1design2.png" align="left" padding:0px width="250px" hspace="20" /><br />
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. <br />
<br><br><br><br><br><br><br><br><br />
</html><br />
<br />
= Results =<br />
----<br />
<br><div align="justify">The result of our lab work was mainly the GATE assembly toolkit and the corresponding vectors. Further experiments were performed to validate the function of the kit both ''in vitro'' and ''in vivo''. <br />
<br />
<br />
== The Toolkit ==<br />
----<br />
<br><br />
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 [[Team:Freiburg/Parts|parts section]] or go to the [http://partsregistry.org Registry of Standard Biological Parts].<br />
<br />
<br />
{|align="center"<br />
|[[Image:AA1sequence.png|400px|no frame|link=]]<br />
|}<br />
<br />
<br />
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.<br />
<br><br><br />
<br />
== Creation of TAL sequences - Golden Gate Cloning ==<br />
----<br />
<br><br />
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<br />
<br />
<br><br />
== Varying Cycle number of GATE assembly has limited effect ==<br />
----<br />
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).<br><br><br />
{|align="center"<br />
|[[Image:colonies.png|400px|no frame|link=]]<br />
|}<br />
<br><br><br />
<br />
== Direpeat Amplification by Colony PCR ==<br />
----<br />
<br><br />
<html><br />
<div align="justify">To assess if the direpeats have indeed been successfully cloned into our expression vector, we have accomplished colony PCR with a variety of samples. To this end, we have designed primers which bind to both ends of the direpeat region and thus amplify the direpeats of our TAL protein. <br />
The original vector contains a kill cassette which kills bacteria unless it is replaced by the direpeats during the Golden Gate Cloning reaction. <br />
This cassette will also be amplified by the designed primers. A distinction between the amplification of the kill cassette and that of direpeats can be made upon the amplicon length: if the kill cassette is amplified, the resulting amplicon will contain 1527bp, while direpeat amplification will produce amplicons of 1276bp length. The difference between these two amplicons is 251bp, and can be detected by agarose gel electrophoresis. (Figure)<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/0/04/Direpeat_amp.jpg" align="right" width="400px" hspace="20" vspace="20" alt="Direpeat amplification by colony PCR"/><br />
<br />
The figure clearly demonstrates the difference in size between the amplicons of negative control (kill cassette still in the vector) and positive (direpeats have replaced the cassette) samples. While lane 2 shows a negative result with a single band of bigger size, all the other samples yielded amplicons of smaller length and thus are considered as positive due to amplification of direpeats.<br />
Nevertheless, it is obvious that the colony PCR did not produce one single product when amplifying the direpeats, but rather a smear consisting of amplicons with varying lengths. <br />
This effect is due to numerous homologies within the direpeats and has previously been described by Briggs et al.<sup>1</sup> . <br />
To eliminate those homologies to the greatest extent possible, we changed codon usage within our direpeats. Nevertheless, as the results of our colony PCR demonstrate, there is still a profound amount of homologies left, which imposes difficulties on the amplification of the direpeats array by PCR and instead results in a smear.<br />
Thus, the existence of this smear indicates the presence of direpeats within the expression vector. Moreover, the light bands at 1276 bp indicates, that the right number of direpeats have been inserted into the vector. We performed 30 colony PCRs from colonies of different GATE assemblies and had no negative results (but in some cases, we could not determine the hight of the light band). Since positive results in colony PCR of TALEs do not exclude wrong order of direpeats, we sequenced 28 clones of different GATE assemblies and analyzed the results: in 27 of the 28 clones, sequencing results entirely matched the right sequence. So the efficiency of GATE assembly is approximately 96 %.<br />
<br><br><br><br />
<br><br />
</html><br />
<br />
== Activation of transcription ==<br />
----<br />
<html><br />
<br><br />
<div align="justify">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).<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/8/84/IGEMres4.png" align="right" width="400px" hspace="20" vspace="20" alt="SEAP essay using the TAL transcription factor plasmid targeting a minimal promotor coupled to a SEAP reporter gene"/><br />
<br />
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 two to photometric analysis.<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
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.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/8/84/IGEMres4.png" width="350px" hspace="20" vspace="20" alt="SEAP assay using the TAL transcription factor plasmid targeting a minimal promotor coupled to a SEAP reporter gene" style="margin-left:170px"/><br />
<br><br />
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).<br />
<br><br />
In the samples, that had been taken 48h after double transfection, the same effects could be demonstrated. <br />
<br><br />
Furthermore, we reapeated the same experiment for a second time. The corresponding data can be viewed here: <html><div style=text-indent:0px><a href="https://static.igem.org/mediawiki/2012/9/9c/Second_Essay.pdf">Second Experiment.</a><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/5/55/TALTF-SEAP-TIME.png" align="middle" width="500px" hspace="20" vspace="20" alt="SEAP assay using the TAL transcription factor plasmid targeting a minimal promotor coupled to a SEAP reporter gene" style="margin-left:120px"/><br />
</html><br />
<br />
== Reference ==<br />
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<br />
<br />
<br><br><br><br />
[[#top|Back to top]]</div>Davsiehttp://2012.igem.org/Team:Freiburg/Project/ExperimentsTeam:Freiburg/Project/Experiments2012-10-23T14:18:00Z<p>Davsie: </p>
<hr />
<div>{{Template:Team:Freiburg}}<br />
__NOTOC__<br />
= Experiments =<br />
----<br />
<br />
<br />
== Gene activation ==<br />
----<br />
<html><br />
<p><br><br />
<br />
<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 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 absorps light at 405 nm and can be measured via photometry. <img src="http://imageshack.us/a/img189/6140/seapplasmid.png" align="right" padding:0px width="450px" hspace="20"/><br />
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.<br />
<br><br><br><br />
</html><br />
<br />
== Experimental design ==<br />
----<br />
<html><br />
<p><br><br />
<img src="http://imageshack.us/a/img268/53/exp1design2.png" align="left" padding:0px width="250px" hspace="20" /><br />
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. <br />
<br><br><br><br><br><br><br><br><br />
</html><br />
<br />
= Results =<br />
----<br />
<br><div align="justify">The result of our lab work was mainly the GATE assembly toolkit and the corresponding vectors. Further experiments were performed to validate the function of the kit both ''in vitro'' and ''in vivo''. <br />
<br />
<br />
== The Toolkit ==<br />
----<br />
<br><br />
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 [[Team:Freiburg/Parts|parts section]] or go to the [http://partsregistry.org Registry of Standard Biological Parts].<br />
<br />
<br />
{|align="center"<br />
|[[Image:AA1sequence.png|400px|no frame|link=]]<br />
|}<br />
<br />
<br />
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.<br />
<br><br><br />
<br />
== Creation of TAL sequences - Golden Gate Cloning ==<br />
----<br />
<br><br />
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<br />
<br />
<br><br />
== Varying Cycle number of GATE assembly has limited effect ==<br />
----<br />
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).<br><br><br />
{|align="center"<br />
|[[Image:colonies.png|400px|no frame|link=]]<br />
|}<br />
<br><br><br />
<br />
== Direpeat Amplification by Colony PCR ==<br />
----<br />
<br><br />
<html><br />
<div align="justify">To assess if the direpeats have indeed been successfully cloned into our expression vector, we have accomplished colony PCR with a variety of samples. To this end, we have designed primers which bind to both ends of the direpeat region and thus amplify the direpeats of our TAL protein. <br />
The original vector contains a kill cassette which kills bacteria unless it is replaced by the direpeats during the Golden Gate Cloning reaction. <br />
This cassette will also be amplified by the designed primers. A distinction between the amplification of the kill cassette and that of direpeats can be made upon the amplicon length: if the kill cassette is amplified, the resulting amplicon will contain 1527bp, while direpeat amplification will produce amplicons of 1276bp length. The difference between these two amplicons is 251bp, and can be detected by agarose gel electrophoresis. (Figure)<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/0/04/Direpeat_amp.jpg" align="right" width="400px" hspace="20" vspace="20" alt="Direpeat amplification by colony PCR"/><br />
<br />
The figure clearly demonstrates the difference in size between the amplicons of negative control (kill cassette still in the vector) and positive (direpeats have replaced the cassette) samples. While lane 2 shows a negative result with a single band of bigger size, all the other samples yielded amplicons of smaller length and thus are considered as positive due to amplification of direpeats.<br />
Nevertheless, it is obvious that the colony PCR did not produce one single product when amplifying the direpeats, but rather a smear consisting of amplicons with varying lengths. <br />
This effect is due to numerous homologies within the direpeats and has previously been described by Briggs et al.<sup>1</sup> . <br />
To eliminate those homologies to the greatest extent possible, we changed codon usage within our direpeats. Nevertheless, as the results of our colony PCR demonstrate, there is still a profound amount of homologies left, which imposes difficulties on the amplification of the direpeats array by PCR and instead results in a smear.<br />
Thus, the existence of this smear indicates the presence of direpeats within the expression vector. Moreover, the light bands at 1276 bp indicates, that the right number of direpeats have been inserted into the vector. We performed 30 colony PCRs from colonies of different GATE assemblies and had no negative results (but in some cases, we could not determine the hight of the light band). Since positive results in colony PCR of TALEs do not exclude wrong order of direpeats, we sequenced 28 clones of different GATE assemblies and analyzed the results: in 27 of the 28 clones, sequencing results entirely matched the right sequence. So the efficiency of GATE assembly is approximately 96 %.<br />
<br><br><br><br />
<br><br />
</html><br />
<br />
== Activation of transcription ==<br />
----<br />
<html><br />
<br><br />
<div align="justify">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).<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/7/73/TALTF-SEAP.png" align="right" width="400px" hspace="20" vspace="20" alt="SEAP essay using the TAL transcription factor plasmid targeting a minimal promotor coupled to a SEAP reporter gene"/><br />
<br />
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 two to photometric analysis.<br />
<br><br><br><br><br><br><br><br><br><br><br><br />
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.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/8/84/IGEMres4.png" width="350px" hspace="20" vspace="20" alt="SEAP assay using the TAL transcription factor plasmid targeting a minimal promotor coupled to a SEAP reporter gene" style="margin-left:170px"/><br />
<br><br />
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).<br />
<br><br />
In the samples, that had been taken 48h after double transfection, the same effects could be demonstrated. <br />
<br><br />
Furthermore, we reapeated the same experiment for a second time. The corresponding data can be viewed here: <html><div style=text-indent:0px><a href="https://static.igem.org/mediawiki/2012/9/9c/Second_Essay.pdf">Second Experiment.</a><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/5/55/TALTF-SEAP-TIME.png" align="middle" width="500px" hspace="20" vspace="20" alt="SEAP assay using the TAL transcription factor plasmid targeting a minimal promotor coupled to a SEAP reporter gene" style="margin-left:120px"/><br />
</html><br />
<br />
== Reference ==<br />
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<br />
<br />
<br><br><br><br />
[[#top|Back to top]]</div>Davsiehttp://2012.igem.org/File:IGEMres4.pngFile:IGEMres4.png2012-10-23T14:17:13Z<p>Davsie: </p>
<hr />
<div></div>Davsiehttp://2012.igem.org/Template:Team:FreiburgTemplate:Team:Freiburg2012-10-19T13:21:31Z<p>Davsie: </p>
<hr />
<div><html><br />
<head><br />
<br />
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height:2px;<br />
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</head></div>Davsiehttp://2012.igem.org/Team:Freiburg/Project/TalTeam:Freiburg/Project/Tal2012-10-14T01:16:14Z<p>Davsie: </p>
<hr />
<div>{{Template:Team:Freiburg}}<br />
__NOTOC__<br />
= Using the Toolkit =<br />
----<br />
<br><br />
<div align="justify">Here, we give you a manual on how to use our toolkit to design TAL proteins. We recommend reading through all of the manual prior to using the toolkit. Also we included a short introductional video on how to you the toolkit.<br />
<br />
<html><br />
<iframe align="center" width="500" height="350" style="margin-left:100px; margin-top:40px ;margin-bottom:50px;"src="http://player.vimeo.com/video/51366791" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe><br />
</html><br />
<br />
= Step 1. Choosing effector and target sequence =<br />
----<br />
<br>First, you need to think about your experimental setup. When working with TAL proteins it's pretty clear you want to target a DNA sequence. To choose your sequence, you need to know some of the operational details of TAL proteins in order to pick it the right way. <br />
<br />
<br />
<b>1. Every TAL binding site starts and ends with a thymine</b><br />
<br />
These thymine binding modules are already inserted in our expression plasmids. So the protein won't bind to other sequences than those which start with a T and end with a T.<br />
<br />
<br />
<b>2. Your sequence must be twelve base pair long</b><br />
<br />
Our toolbox is optimized for sequences of twelve plus two (the thymine at upstream and downstream positions). This lenght guarantees a high specifity and a library size that's good to handle at the same time.<br />
<br />
<br />
You can check out the following online softwares for perfect TAL-TF or TALEN binding sites:<br><br><br />
https://boglab.plp.iastate.edu/node/add/talen (for TALENs)<br><br />
https://boglab.plp.iastate.edu/node/add/single-tale (for TAL-TFs)<br />
<br />
<br><br />
<br />
<br><br />
<br />
= Step 2. Building a TAL =<br />
----<br />
<br>Building your TAL starts with your selected sequence. In this manual, we use a fictive sequence that you can substitute with your own. <p>Our sequence will be as follows:</p><br />
<br />
<br />
[[Image:sequence1.png|200px|center|no frame]]<br />
<br />
<br />
<br />
<br>Because the two thymines are already in the cloning vector, they are of no interest for our TAL protein:<br />
<br />
[[Image:sequence2.png|200px|center|no frame]]<br />
<br />
<br />
<br>To build this sequence from our toolkit we need to split it up in pairs of two:<br />
<br />
<br />
[[Image:sequence3.png|350px|center|no frame]]<br />
<br />
<br>Now we need to give our pairs position numbers inside the TAL protein:<br />
<br />
<br />
[[Image:sequence4.png|500px|center|no frame]]<br />
<br />
<br />
<br />
<br>Now we can start taking the parts out of the toolkit. A short look at the toolkit shows you that for every possible pair of bases, for example AA, we have 6 places. Every place stands for one of the six possible positions of the pair AA inside the TAL protein.<br />
<br />
<br />
[[Image:toolkit3.png|300px|center|no frame]]<br />
<br />
<br />
<br>All you have to do now is pick the six direpeats consistent with the six pairs of your sequence. In our case, we would take the the first one of AA because the first pair of bases in our sequence is AA. Then we take the second one of TG the third of AG and so forth. The idea behind this is that every direpeat knows through his downstream and upstream part at which position of the final TAL protein it ought to be. You can find the exact mechanisms behind this in the [[Team:Freiburg/Project/Overview|'GATE Assembly Kit']] part of our project section. <br><br><br />
<br />
[[Image:sequence5.png|500px|center|no frame]]<br />
<br />
<br />
<br><br />
For lazy iGEM students, we have written a simple program. So you just have to type in your target DNA sequence and we give you a list of parts that you need to pipet into your Golden Gate reaction mix:<br><br><br />
<br />
http://omnibus.uni-freiburg.de/~lb125<br />
<br />
<html><br />
<iframe src="http://omnibus.uni-freiburg.de/~lb125/index.html"; width=770px; height=170px><br />
</iframe><br />
</html><br />
<br />
<br><br />
<br />
= Step 3. Adding a Function =<br />
----<br />
<br><br />
Now that you have your TAL biobricks, you are almost done. But targeting a sequence without doing anything is not really helpful, so you need a fusion protein that does something to your DNA. There are a couple of things you could do with your target sequence, and normally you have thought of this before you chose your sequence. With our toolkit you get a transcription factor to turn on or enhance the trancription of a gene and a restriction enzyme to make cuts wherever you want. Every one of these factors is already placed inside the final TAL vector and designed to fit the 3'-end of your TAL biobricks. Conveniently, you just choose one and put it in your reaction tube along with the other biobricks.<br />
<br />
<br />
<br />
[[Image:TALfunction.png|600px|center|no frame]]<br />
<br />
<br />
<br />
With the six TAL biobricks and the fusion enzyme in your reaction tube you now only need the type two restriction enzyme BsmB1 and a T7 Ligase to put all the parts together.<br><br><br />
<br />
[[Image:protocolggc.png|300px|left|no frame]]<br />
<br />
<br><br />
<br />
[[Image:thermocycler.png|200px|center|no frame]]<br />
<br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
= Step 4. Transformation and Use =<br />
----<br />
<br><br />
Transform 5 μl of the GATE assembly product into 50 μl of transformation competent bacteria.<br> <br />
<br>'''Important note:''' Your cells need to be sensitive to the ccdB kill cassette in our TAL expression vectors! Otherwise also bacteria that have taken up plasmids without the six direpeats will form false positive colonies. We used the DH10B E.coli strain.<br><br />
In case you want to express your TALE in bacteria, you need to induce the promoter of our prokaryotic expression plasmid with IPTG. <br>For use in a eukaryotic system, such as HEK 239 cells, perform a midiprep and directly transfect the eukaryotic TAL expression plasmid (or its derivatives pTAL-TF, pTALEN etc.) according to your transfection protocol. <br />
<!--- The Mission, Experiments ---><br />
<br><br><br><br><br><br><br />
[[#top|Back to top]]</div>Davsiehttp://2012.igem.org/Team:Freiburg/Project/TalTeam:Freiburg/Project/Tal2012-10-14T01:15:35Z<p>Davsie: </p>
<hr />
<div>{{Template:Team:Freiburg}}<br />
__NOTOC__<br />
= Using the Toolkit =<br />
----<br />
<br><br />
<div align="justify">Here, we give you a manual on how to use our toolkit to design TAL proteins. We recommend reading through all of the manual prior to using the toolkit. Also we included a short introductional video on how to you the toolkit.<br />
<br />
<html><br />
<iframe align="center" width="500" height="350" style="margin-left:100px; margin-top:40px ;margin-bottom:50px;"src="http://player.vimeo.com/video/51366791" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe><br />
</html><br />
<br />
= Step 1. Choosing effector and target sequence =<br />
----<br />
<br>First, you need to think about your experimental setup. When working with TAL proteins it's pretty clear you want to target a DNA sequence. To choose your sequence, you need to know some of the operational details of TAL proteins in order to pick it the right way. <br />
<br />
<br />
<b>1. Every TAL binding site starts and ends with a thymine</b><br />
<br />
These thymine binding modules are already inserted in our expression plasmids. So the protein won't bind to other sequences than those which start with a T and end with a T.<br />
<br />
<br />
<b>2. Your sequence must be twelve base pair long</b><br />
<br />
Our toolbox is optimized for sequences of twelve plus two (the thymine at upstream and downstream positions). This lenght guarantees a high specifity and a library size that's good to handle at the same time.<br />
<br />
<br />
You can check out the following online softwares for perfect TAL-TF or TALEN binding sites:<br><br><br />
https://boglab.plp.iastate.edu/node/add/talen (for TALENs)<br><br />
https://boglab.plp.iastate.edu/node/add/single-tale (for TAL-TFs)<br />
<br />
<br><br />
<br />
<br><br />
<br />
= Step 2. Building a TAL =<br />
----<br />
<br>Building your TAL starts with your selected sequence. In this manual, we use a fictive sequence that you can substitute with your own. <p>Our sequence will be as follows:</p><br />
<br />
<br />
[[Image:sequence1.png|200px|center|no frame]]<br />
<br />
<br />
<br />
<br>Because the two thymines are already in the cloning vector, they are of no interest for our TAL protein:<br />
<br />
[[Image:sequence2.png|200px|center|no frame]]<br />
<br />
<br />
<br>To build this sequence from our toolkit we need to split it up in pairs of two:<br />
<br />
<br />
[[Image:sequence3.png|350px|center|no frame]]<br />
<br />
<br>Now we need to give our pairs position numbers inside the TAL protein:<br />
<br />
<br />
[[Image:sequence4.png|500px|center|no frame]]<br />
<br />
<br />
<br />
<br>Now we can start taking the parts out of the toolkit. A short look at the toolkit shows you that for every possible pair of bases, for example AA, we have 6 places. Every place stands for one of the six possible positions of the pair AA inside the TAL protein.<br />
<br />
<br />
[[Image:toolkit3.png|300px|center|no frame]]<br />
<br />
<br />
<br>All you have to do now is pick the six direpeats consistent with the six pairs of your sequence. In our case, we would take the the first one of AA because the first pair of bases in our sequence is AA. Then we take the second one of TG the third of AG and so forth. The idea behind this is that every direpeat knows through his downstream and upstream part at which position of the final TAL protein it ought to be. You can find the exact mechanisms behind this in the [[Team:Freiburg/Project/Overview|'GATE Assembly Kit']] part of our project section. <br><br><br />
<br />
[[Image:sequence5.png|500px|center|no frame]]<br />
<br />
<br />
<br><br />
For lazy iGEM students, we have written a simple program. So you just have to type in your target DNA sequence and we give you a list of parts that you need to pipet into your Golden Gate reaction mix:<br><br><br />
<br />
http://omnibus.uni-freiburg.de/~lb125<br />
<br />
<html><br />
<iframe src="http://omnibus.uni-freiburg.de/~lb125/index.html"; width=770px; height=150px><br />
</iframe><br />
</html><br />
<br />
<br><br />
<br />
= Step 3. Adding a Function =<br />
----<br />
<br><br />
Now that you have your TAL biobricks, you are almost done. But targeting a sequence without doing anything is not really helpful, so you need a fusion protein that does something to your DNA. There are a couple of things you could do with your target sequence, and normally you have thought of this before you chose your sequence. With our toolkit you get a transcription factor to turn on or enhance the trancription of a gene and a restriction enzyme to make cuts wherever you want. Every one of these factors is already placed inside the final TAL vector and designed to fit the 3'-end of your TAL biobricks. Conveniently, you just choose one and put it in your reaction tube along with the other biobricks.<br />
<br />
<br />
<br />
[[Image:TALfunction.png|600px|center|no frame]]<br />
<br />
<br />
<br />
With the six TAL biobricks and the fusion enzyme in your reaction tube you now only need the type two restriction enzyme BsmB1 and a T7 Ligase to put all the parts together.<br><br><br />
<br />
[[Image:protocolggc.png|300px|left|no frame]]<br />
<br />
<br><br />
<br />
[[Image:thermocycler.png|200px|center|no frame]]<br />
<br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
= Step 4. Transformation and Use =<br />
----<br />
<br><br />
Transform 5 μl of the GATE assembly product into 50 μl of transformation competent bacteria.<br> <br />
<br>'''Important note:''' Your cells need to be sensitive to the ccdB kill cassette in our TAL expression vectors! Otherwise also bacteria that have taken up plasmids without the six direpeats will form false positive colonies. We used the DH10B E.coli strain.<br><br />
In case you want to express your TALE in bacteria, you need to induce the promoter of our prokaryotic expression plasmid with IPTG. <br>For use in a eukaryotic system, such as HEK 239 cells, perform a midiprep and directly transfect the eukaryotic TAL expression plasmid (or its derivatives pTAL-TF, pTALEN etc.) according to your transfection protocol. <br />
<!--- The Mission, Experiments ---><br />
<br><br><br><br><br><br><br />
[[#top|Back to top]]</div>Davsiehttp://2012.igem.org/Team:Freiburg/Project/TalTeam:Freiburg/Project/Tal2012-10-14T01:14:57Z<p>Davsie: </p>
<hr />
<div>{{Template:Team:Freiburg}}<br />
__NOTOC__<br />
= Using the Toolkit =<br />
----<br />
<br><br />
<div align="justify">Here, we give you a manual on how to use our toolkit to design TAL proteins. We recommend reading through all of the manual prior to using the toolkit. Also we included a short introductional video on how to you the toolkit.<br />
<br />
<html><br />
<iframe align="center" width="500" height="350" style="margin-left:100px; margin-top:40px ;margin-bottom:50px;"src="http://player.vimeo.com/video/51366791" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe><br />
</html><br />
<br />
= Step 1. Choosing effector and target sequence =<br />
----<br />
<br>First, you need to think about your experimental setup. When working with TAL proteins it's pretty clear you want to target a DNA sequence. To choose your sequence, you need to know some of the operational details of TAL proteins in order to pick it the right way. <br />
<br />
<br />
<b>1. Every TAL binding site starts and ends with a thymine</b><br />
<br />
These thymine binding modules are already inserted in our expression plasmids. So the protein won't bind to other sequences than those which start with a T and end with a T.<br />
<br />
<br />
<b>2. Your sequence must be twelve base pair long</b><br />
<br />
Our toolbox is optimized for sequences of twelve plus two (the thymine at upstream and downstream positions). This lenght guarantees a high specifity and a library size that's good to handle at the same time.<br />
<br />
<br />
You can check out the following online softwares for perfect TAL-TF or TALEN binding sites:<br><br><br />
https://boglab.plp.iastate.edu/node/add/talen (for TALENs)<br><br />
https://boglab.plp.iastate.edu/node/add/single-tale (for TAL-TFs)<br />
<br />
<br><br />
<br />
<br><br />
<br />
= Step 2. Building a TAL =<br />
----<br />
<br>Building your TAL starts with your selected sequence. In this manual, we use a fictive sequence that you can substitute with your own. <p>Our sequence will be as follows:</p><br />
<br />
<br />
[[Image:sequence1.png|200px|center|no frame]]<br />
<br />
<br />
<br />
<br>Because the two thymines are already in the cloning vector, they are of no interest for our TAL protein:<br />
<br />
[[Image:sequence2.png|200px|center|no frame]]<br />
<br />
<br />
<br>To build this sequence from our toolkit we need to split it up in pairs of two:<br />
<br />
<br />
[[Image:sequence3.png|350px|center|no frame]]<br />
<br />
<br>Now we need to give our pairs position numbers inside the TAL protein:<br />
<br />
<br />
[[Image:sequence4.png|500px|center|no frame]]<br />
<br />
<br />
<br />
<br>Now we can start taking the parts out of the toolkit. A short look at the toolkit shows you that for every possible pair of bases, for example AA, we have 6 places. Every place stands for one of the six possible positions of the pair AA inside the TAL protein.<br />
<br />
<br />
[[Image:toolkit3.png|300px|center|no frame]]<br />
<br />
<br />
<br>All you have to do now is pick the six direpeats consistent with the six pairs of your sequence. In our case, we would take the the first one of AA because the first pair of bases in our sequence is AA. Then we take the second one of TG the third of AG and so forth. The idea behind this is that every direpeat knows through his downstream and upstream part at which position of the final TAL protein it ought to be. You can find the exact mechanisms behind this in the [[Team:Freiburg/Project/Overview|'GATE Assembly Kit']] part of our project section. <br><br><br />
<br />
[[Image:sequence5.png|500px|center|no frame]]<br />
<br />
<br />
<br><br />
For lazy iGEM students, we have written a simple program. So you just have to type in your target DNA sequence and we give you a list of parts that you need to pipet into your Golden Gate reaction mix:<br><br><br />
<br />
http://omnibus.uni-freiburg.de/~lb125<br />
<br />
<html><br />
<iframe src="http://omnibus.uni-freiburg.de/~lb125/index.html"; width=770px; height=200px><br />
</iframe><br />
</html><br />
<br />
<br><br />
<br />
= Step 3. Adding a Function =<br />
----<br />
<br><br />
Now that you have your TAL biobricks, you are almost done. But targeting a sequence without doing anything is not really helpful, so you need a fusion protein that does something to your DNA. There are a couple of things you could do with your target sequence, and normally you have thought of this before you chose your sequence. With our toolkit you get a transcription factor to turn on or enhance the trancription of a gene and a restriction enzyme to make cuts wherever you want. Every one of these factors is already placed inside the final TAL vector and designed to fit the 3'-end of your TAL biobricks. Conveniently, you just choose one and put it in your reaction tube along with the other biobricks.<br />
<br />
<br />
<br />
[[Image:TALfunction.png|600px|center|no frame]]<br />
<br />
<br />
<br />
With the six TAL biobricks and the fusion enzyme in your reaction tube you now only need the type two restriction enzyme BsmB1 and a T7 Ligase to put all the parts together.<br><br><br />
<br />
[[Image:protocolggc.png|300px|left|no frame]]<br />
<br />
<br><br />
<br />
[[Image:thermocycler.png|200px|center|no frame]]<br />
<br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
= Step 4. Transformation and Use =<br />
----<br />
<br><br />
Transform 5 μl of the GATE assembly product into 50 μl of transformation competent bacteria.<br> <br />
<br>'''Important note:''' Your cells need to be sensitive to the ccdB kill cassette in our TAL expression vectors! Otherwise also bacteria that have taken up plasmids without the six direpeats will form false positive colonies. We used the DH10B E.coli strain.<br><br />
In case you want to express your TALE in bacteria, you need to induce the promoter of our prokaryotic expression plasmid with IPTG. <br>For use in a eukaryotic system, such as HEK 239 cells, perform a midiprep and directly transfect the eukaryotic TAL expression plasmid (or its derivatives pTAL-TF, pTALEN etc.) according to your transfection protocol. <br />
<!--- The Mission, Experiments ---><br />
<br><br><br><br><br><br><br />
[[#top|Back to top]]</div>Davsiehttp://2012.igem.org/Team:Freiburg/Project/TalTeam:Freiburg/Project/Tal2012-10-14T01:10:48Z<p>Davsie: </p>
<hr />
<div>{{Template:Team:Freiburg}}<br />
__NOTOC__<br />
= Using the Toolkit =<br />
----<br />
<br><br />
<div align="justify">Here, we give you a manual on how to use our toolkit to design TAL proteins. We recommend reading through all of the manual prior to using the toolkit. Also we included a short introductional video on how to you the toolkit.<br />
<br />
<html><br />
<iframe align="center" width="500" height="350" style="margin-left:100px; margin-top:40px ;margin-bottom:50px;"src="http://player.vimeo.com/video/51366791" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe><br />
</html><br />
<br />
= Step 1. Choosing effector and target sequence =<br />
----<br />
<br>First, you need to think about your experimental setup. When working with TAL proteins it's pretty clear you want to target a DNA sequence. To choose your sequence, you need to know some of the operational details of TAL proteins in order to pick it the right way. <br />
<br />
<br />
<b>1. Every TAL binding site starts and ends with a thymine</b><br />
<br />
These thymine binding modules are already inserted in our expression plasmids. So the protein won't bind to other sequences than those which start with a T and end with a T.<br />
<br />
<br />
<b>2. Your sequence must be twelve base pair long</b><br />
<br />
Our toolbox is optimized for sequences of twelve plus two (the thymine at upstream and downstream positions). This lenght guarantees a high specifity and a library size that's good to handle at the same time.<br />
<br />
<br />
You can check out the following online softwares for perfect TAL-TF or TALEN binding sites:<br><br><br />
https://boglab.plp.iastate.edu/node/add/talen (for TALENs)<br><br />
https://boglab.plp.iastate.edu/node/add/single-tale (for TAL-TFs)<br />
<br />
<br><br />
<br />
<br><br />
<br />
= Step 2. Building a TAL =<br />
----<br />
<br>Building your TAL starts with your selected sequence. In this manual, we use a fictive sequence that you can substitute with your own. <p>Our sequence will be as follows:</p><br />
<br />
<br />
[[Image:sequence1.png|200px|center|no frame]]<br />
<br />
<br />
<br />
<br>Because the two thymines are already in the cloning vector, they are of no interest for our TAL protein:<br />
<br />
[[Image:sequence2.png|200px|center|no frame]]<br />
<br />
<br />
<br>To build this sequence from our toolkit we need to split it up in pairs of two:<br />
<br />
<br />
[[Image:sequence3.png|350px|center|no frame]]<br />
<br />
<br>Now we need to give our pairs position numbers inside the TAL protein:<br />
<br />
<br />
[[Image:sequence4.png|500px|center|no frame]]<br />
<br />
<br />
<br />
<br>Now we can start taking the parts out of the toolkit. A short look at the toolkit shows you that for every possible pair of bases, for example AA, we have 6 places. Every place stands for one of the six possible positions of the pair AA inside the TAL protein.<br />
<br />
<br />
[[Image:toolkit3.png|300px|center|no frame]]<br />
<br />
<br />
<br>All you have to do now is pick the six direpeats consistent with the six pairs of your sequence. In our case, we would take the the first one of AA because the first pair of bases in our sequence is AA. Then we take the second one of TG the third of AG and so forth. The idea behind this is that every direpeat knows through his downstream and upstream part at which position of the final TAL protein it ought to be. You can find the exact mechanisms behind this in the [[Team:Freiburg/Project/Overview|'GATE Assembly Kit']] part of our project section. <br><br><br />
<br />
[[Image:sequence5.png|500px|center|no frame]]<br />
<br />
<br />
<br><br />
For lazy iGEM students, we have written a simple program. So you just have to type in your target DNA sequence and we give you a list of parts that you need to pipet into your Golden Gate reaction mix:<br><br><br />
<br />
http://omnibus.uni-freiburg.de/~lb125<br />
<br />
<html><br />
<iframe src="http://omnibus.uni-freiburg.de/~lb125/index.html"; width=570px; height=200px><br />
</iframe><br />
</html><br />
<br />
<br><br />
<br />
= Step 3. Adding a Function =<br />
----<br />
<br><br />
Now that you have your TAL biobricks, you are almost done. But targeting a sequence without doing anything is not really helpful, so you need a fusion protein that does something to your DNA. There are a couple of things you could do with your target sequence, and normally you have thought of this before you chose your sequence. With our toolkit you get a transcription factor to turn on or enhance the trancription of a gene and a restriction enzyme to make cuts wherever you want. Every one of these factors is already placed inside the final TAL vector and designed to fit the 3'-end of your TAL biobricks. Conveniently, you just choose one and put it in your reaction tube along with the other biobricks.<br />
<br />
<br />
<br />
[[Image:TALfunction.png|600px|center|no frame]]<br />
<br />
<br />
<br />
With the six TAL biobricks and the fusion enzyme in your reaction tube you now only need the type two restriction enzyme BsmB1 and a T7 Ligase to put all the parts together.<br><br><br />
<br />
[[Image:protocolggc.png|300px|left|no frame]]<br />
<br />
<br><br />
<br />
[[Image:thermocycler.png|200px|center|no frame]]<br />
<br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
= Step 4. Transformation and Use =<br />
----<br />
<br><br />
Transform 5 μl of the GATE assembly product into 50 μl of transformation competent bacteria.<br> <br />
<br>'''Important note:''' Your cells need to be sensitive to the ccdB kill cassette in our TAL expression vectors! Otherwise also bacteria that have taken up plasmids without the six direpeats will form false positive colonies. We used the DH10B E.coli strain.<br><br />
In case you want to express your TALE in bacteria, you need to induce the promoter of our prokaryotic expression plasmid with IPTG. <br>For use in a eukaryotic system, such as HEK 239 cells, perform a midiprep and directly transfect the eukaryotic TAL expression plasmid (or its derivatives pTAL-TF, pTALEN etc.) according to your transfection protocol. <br />
<!--- The Mission, Experiments ---><br />
<br><br><br><br><br><br><br />
[[#top|Back to top]]</div>Davsiehttp://2012.igem.org/Team:Freiburg/Project/TalTeam:Freiburg/Project/Tal2012-10-14T01:07:33Z<p>Davsie: </p>
<hr />
<div>{{Template:Team:Freiburg}}<br />
__NOTOC__<br />
= Using the Toolkit =<br />
----<br />
<br><br />
<div align="justify">Here, we give you a manual on how to use our toolkit to design TAL proteins. We recommend reading through all of the manual prior to using the toolkit.<br />
<br />
<html><br />
<iframe align="center" width="500" height="350" style="margin-left:100px; margin-top:40px ;margin-bottom:50px;"src="http://player.vimeo.com/video/51366791" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe><br />
</html><br />
<br />
= Step 1. Choosing effector and target sequence =<br />
----<br />
<br>First, you need to think about your experimental setup. When working with TAL proteins it's pretty clear you want to target a DNA sequence. To choose your sequence, you need to know some of the operational details of TAL proteins in order to pick it the right way. <br />
<br />
<br />
<b>1. Every TAL binding site starts and ends with a thymine</b><br />
<br />
These thymine binding modules are already inserted in our expression plasmids. So the protein won't bind to other sequences than those which start with a T and end with a T.<br />
<br />
<br />
<b>2. Your sequence must be twelve base pair long</b><br />
<br />
Our toolbox is optimized for sequences of twelve plus two (the thymine at upstream and downstream positions). This lenght guarantees a high specifity and a library size that's good to handle at the same time.<br />
<br />
<br />
You can check out the following online softwares for perfect TAL-TF or TALEN binding sites:<br><br><br />
https://boglab.plp.iastate.edu/node/add/talen (for TALENs)<br><br />
https://boglab.plp.iastate.edu/node/add/single-tale (for TAL-TFs)<br />
<br />
<br><br />
<br />
<br><br />
<br />
= Step 2. Building a TAL =<br />
----<br />
<br>Building your TAL starts with your selected sequence. In this manual, we use a fictive sequence that you can substitute with your own. <p>Our sequence will be as follows:</p><br />
<br />
<br />
[[Image:sequence1.png|200px|center|no frame]]<br />
<br />
<br />
<br />
<br>Because the two thymines are already in the cloning vector, they are of no interest for our TAL protein:<br />
<br />
[[Image:sequence2.png|200px|center|no frame]]<br />
<br />
<br />
<br>To build this sequence from our toolkit we need to split it up in pairs of two:<br />
<br />
<br />
[[Image:sequence3.png|350px|center|no frame]]<br />
<br />
<br>Now we need to give our pairs position numbers inside the TAL protein:<br />
<br />
<br />
[[Image:sequence4.png|500px|center|no frame]]<br />
<br />
<br />
<br />
<br>Now we can start taking the parts out of the toolkit. A short look at the toolkit shows you that for every possible pair of bases, for example AA, we have 6 places. Every place stands for one of the six possible positions of the pair AA inside the TAL protein.<br />
<br />
<br />
[[Image:toolkit3.png|300px|center|no frame]]<br />
<br />
<br />
<br>All you have to do now is pick the six direpeats consistent with the six pairs of your sequence. In our case, we would take the the first one of AA because the first pair of bases in our sequence is AA. Then we take the second one of TG the third of AG and so forth. The idea behind this is that every direpeat knows through his downstream and upstream part at which position of the final TAL protein it ought to be. You can find the exact mechanisms behind this in the [[Team:Freiburg/Project/Overview|'GATE Assembly Kit']] part of our project section. <br><br><br />
<br />
[[Image:sequence5.png|500px|center|no frame]]<br />
<br />
<br />
<br><br />
For lazy iGEM students, we have written a simple program. So you just have to type in your target DNA sequence and we give you a list of parts that you need to pipet into your Golden Gate reaction mix:<br><br><br />
<br />
http://omnibus.uni-freiburg.de/~lb125<br />
<br />
<html><br />
<iframe src="http://omnibus.uni-freiburg.de/~lb125/index.html"; width=570px; height=200px><br />
</iframe><br />
</html><br />
<br />
<br><br />
<br />
= Step 3. Adding a Function =<br />
----<br />
<br><br />
Now that you have your TAL biobricks, you are almost done. But targeting a sequence without doing anything is not really helpful, so you need a fusion protein that does something to your DNA. There are a couple of things you could do with your target sequence, and normally you have thought of this before you chose your sequence. With our toolkit you get a transcription factor to turn on or enhance the trancription of a gene and a restriction enzyme to make cuts wherever you want. Every one of these factors is already placed inside the final TAL vector and designed to fit the 3'-end of your TAL biobricks. Conveniently, you just choose one and put it in your reaction tube along with the other biobricks.<br />
<br />
<br />
<br />
[[Image:TALfunction.png|600px|center|no frame]]<br />
<br />
<br />
<br />
With the six TAL biobricks and the fusion enzyme in your reaction tube you now only need the type two restriction enzyme BsmB1 and a T7 Ligase to put all the parts together.<br><br><br />
<br />
[[Image:protocolggc.png|300px|left|no frame]]<br />
<br />
<br><br />
<br />
[[Image:thermocycler.png|200px|center|no frame]]<br />
<br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
= Step 4. Transformation and Use =<br />
----<br />
<br><br />
Transform 5 μl of the GATE assembly product into 50 μl of transformation competent bacteria.<br> <br />
<br>'''Important note:''' Your cells need to be sensitive to the ccdB kill cassette in our TAL expression vectors! Otherwise also bacteria that have taken up plasmids without the six direpeats will form false positive colonies. We used the DH10B E.coli strain.<br><br />
In case you want to express your TALE in bacteria, you need to induce the promoter of our prokaryotic expression plasmid with IPTG. <br>For use in a eukaryotic system, such as HEK 239 cells, perform a midiprep and directly transfect the eukaryotic TAL expression plasmid (or its derivatives pTAL-TF, pTALEN etc.) according to your transfection protocol. <br />
<!--- The Mission, Experiments ---><br />
<br><br><br><br><br><br><br />
[[#top|Back to top]]</div>Davsie