Team:Freiburg/Project/Tal

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=<span style="color:#2244AA"> Using the Toolkit =
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= Using the Toolkit =
 +
----
 +
<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. Moreover, we included a short introductional video on how to use the toolkit.
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<html>
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<br><br>
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<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>
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<br><br><br><br>
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</html>
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= Step 1. Choosing effector and target sequence =
----
----
 +
<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.
-
<div align="justify">Here we give you a short manual on how to use our toolkit to design TAL poteins. We recommend reading through all of the manual first prior to toolkit use.
 
-
=<span style="color:#2244AA"> Step 1. The Experiment =
+
<b>1. Every TAL binding site starts and ends with a thymine</b>
-
First you need to think about your experiment and what you want to do, when working with TAL proteins its pretty clear you want to target a dna sequence. To choose your sequence you need to know some of the limitations of TAL proteins to pick it the right way.  
+
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.
-
<b>1. Every TAL protein starts and ends with a Thymin</b>
+
<b>2. Your sequence must be twelve base pair long</b>
-
This thymin is already inserted in front of your TAL protein when you use our toolkit but it should also be in front of the first base of your sequence
+
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.
-
<b>2. Your sequence must be twelve bases long</b>
+
You can check out the following online softwares for perfect TAL-TF or TALEN binding sites:<br><br>
 +
https://boglab.plp.iastate.edu/node/add/talen (for TALENs)<br>
 +
https://boglab.plp.iastate.edu/node/add/single-tale (for TAL-TFs)
-
Our Toolbox is optimized for sequences of twelve plus two (the thymin at upstream and downstrem) this lenght guarantes a high enough specifity and at the same time a librarysize that good to handle.
+
<br>
 +
<br>
-
If you found a sequence that suits these limitations you can start building your TAL protein.
+
= Step 2. Building a TAL =
 +
----
 +
<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>
-
=<span style="color:#2244AA"> Step 2. Building a TAL =
 
-
For building your TAL you start with your selected sequence, in this manual we use a fictive sequence that you can substitute with your own. <p>Our sequence will be:</p>
+
[[Image:sequence1.png|200px|center|no frame|link=]]
-
{|align="left"
 
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|[[Image:sequence1.png|200px|no frame]]
 
-
|}
 
-
<br><br>Because the two thymins are already in the cloning vector they are not interesting for our TAL protein:
+
<br>Because the two thymines are already in the cloning vector, they are of no interest for our TAL protein:
-
{|align="left"
+
[[Image:sequence2.png|200px|center|no frame|link=]]
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|[[Image:sequence2.png|200px|no frame]]
+
-
|}
+
-
<br><br>To build this sequence from our toolkit we need to split it up in pairs of two:
+
<br>To build this sequence from our toolkit we need to split it up in pairs of two:
-
{|align="left"
 
-
|[[Image:sequence3.png|350px|no frame]]
 
-
|}
 
 +
[[Image:sequence3.png|350px|center|no frame|link=]]
-
<br><br>Now we need to give our pairs position numbers inside the TAL protein:
+
<br>Now, we need to give our pairs position numbers inside the TAL protein:
-
{|align="left"
 
-
|[[Image:sequence4.png|500px|no frame]]
 
-
|}
 
 +
[[Image:sequence4.png|500px|center|no frame|link=]]
-
<br><br>Now we can start taking the parts out of the toolkit. A short look at the toolkit shows you that for every posible pair of bases like for example AA we have 6 places. Every place stands for one of the six possiple positions of the pair AA inside the TAL protein.
 
-
{|align="center"
 
-
|[[Image:toolkit3.png|300px|no frame]]
 
-
|}
 
-
<br>All you have to do now is pick the six direpeats consistend with the six pairs of bases 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 that every direpeat knows through his downstream and upstream part at which position of the final TAL protein it belongs. You can find the exact theory behind this in the "Building a toolkit" part in the project section of this wiki.
+
<br>Next, 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.
-
{|align="center"
 
-
|[[Image:sequence5.png|500px|no frame]]
 
-
|}
 
-
=<span style="color:#2244AA"> Step 3. Adding a Function =
+
[[Image:toolkit3.png|300px|center|no frame|link=]]
-
Now that you have your TAL biobricks you a almost ready to start working. But targeting a sequence without doing anything is not really helpfull, 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 choose your sequence. With our toolkit you get a transcriptionfactor to turn on or enhance the trancription of a gen, a transcriptionfactor to turn down or off the transcription and several other factors like epigeneticmodificators. Every one of these factors is already insde the final TAL vektor and designed to fit at the 3'-end of your TAL biobricks, so you just have to choose one and put it in your reaction tube to the other biobricks.
+
<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>
 +
[[Image:sequence5.png|500px|center|no frame|link=]]
-
{|align="center"
 
-
|[[Image:TALfunction.png|600px|no frame]]
 
-
|}
 
 +
<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>
-
With the six TAL biobricks and the fusion enzyme in your reaction tube you now only need the typ two restriction enzyme BsmB1 and a T7 Ligase to put all the parts together.
 
 +
<html>
 +
<div align="center">
 +
<iframe src="http://omnibus.uni-freiburg.de/~lb125/index.html"; width=95%; frameborder="0"; scrolling="no">
 +
</iframe>
 +
</div>
 +
</html>
 +
 +
 +
 +
<br>
 +
 +
= Step 3. Adding a Function =
 +
----
 +
<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), a restriction enzyme (to make cuts wherever you want) and a desaminase (to make site-specific mutations). 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.
 +
 +
 +
 +
[[Image:TALfunction.png|600px|center|no frame|link=]]
 +
 +
 +
 +
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>
 +
 +
[[Image:protocolggc.png|300px|left|no frame|link=]]
 +
 +
<br>
 +
 +
[[Image:thermocycler.png|200px|center|no frame|link=]]
 +
 +
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
 +
= Step 4. Transformation and Use =
 +
----
 +
<br>
 +
Transform 5 μl of the GATE assembly product into 50 μl of transformation competent bacteria.<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>
 +
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.
<!--- The Mission, Experiments --->
<!--- The Mission, Experiments --->
 +
<br><br><br><br><br><br>
 +
[[#top|Back to top]]

Latest revision as of 01:15, 27 October 2012




Using the Toolkit



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. Moreover, we included a short introductional video on how to use the toolkit.







Step 1. Choosing effector and target sequence



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.


1. Every TAL binding site starts and ends with a thymine

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.


2. Your sequence must be twelve base pair long

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.


You can check out the following online softwares for perfect TAL-TF or TALEN binding sites:

https://boglab.plp.iastate.edu/node/add/talen (for TALENs)
https://boglab.plp.iastate.edu/node/add/single-tale (for TAL-TFs)



Step 2. Building a TAL



Building your TAL starts with your selected sequence. In this manual, we use a fictive sequence that you can substitute with your own.

Our sequence will be as follows:


no frame



Because the two thymines are already in the cloning vector, they are of no interest for our TAL protein:

no frame



To build this sequence from our toolkit we need to split it up in pairs of two:


no frame


Now, we need to give our pairs position numbers inside the TAL protein:


no frame



Next, 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.


no frame



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 'GATE Assembly Kit' part of our project section.

no frame



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:




Step 3. Adding a Function



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), a restriction enzyme (to make cuts wherever you want) and a desaminase (to make site-specific mutations). 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.


no frame


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.

no frame


no frame






Step 4. Transformation and Use



Transform 5 μl of the GATE assembly product into 50 μl of transformation competent bacteria.

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.
In case you want to express your TALE in bacteria, you need to induce the promoter of our prokaryotic expression plasmid with IPTG.
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.





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