Team:Tsinghua-A/Project/Design

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     <h2 class="textTitle" style="margin-top:135px;">What is ‘programmable‘?</h2>
     <h2 class="textTitle" style="margin-top:135px;">What is ‘programmable‘?</h2>
<p> Logic, which tends to be presented in the form of truth tables, shows a relationship between inputs and outputs. Below are the truth tables of the two basic gates: the And gate and the Or gate. So we quickly find the difference lies in the outputs when only one of the inputs is true. In the And gate if there are only one true input, either A or B, the output is false. But in the Or gate it’s to the contrary. So if we can control the output when only one input is true, we can successfully make the switch between the And gate and the Or gate, which can be called a programmable device.
<p> Logic, which tends to be presented in the form of truth tables, shows a relationship between inputs and outputs. Below are the truth tables of the two basic gates: the And gate and the Or gate. So we quickly find the difference lies in the outputs when only one of the inputs is true. In the And gate if there are only one true input, either A or B, the output is false. But in the Or gate it’s to the contrary. So if we can control the output when only one input is true, we can successfully make the switch between the And gate and the Or gate, which can be called a programmable device.
 +
 +
<table border="1" align="center">
 +
<col width="300">
 +
<col width="300">
 +
 +
<tr>
 +
<th> AND Gate</th>
 +
<th> OR Gate</th>
 +
 +
 +
</tr>
 +
 +
<tr>
 +
<td> <table border="1" align="center">
 +
<col width="100">
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<col width="100">
 +
<col width="100">
 +
 +
<tr>
 +
<th> A</th>
 +
<th> B</th>
 +
<th> Output </th>
 +
 +
</tr>
 +
 +
<tr>
 +
<td> 0 </td>
 +
<td> 0 </td>
 +
<td> 0 </td>
 +
 +
</tr>
 +
 +
<tr>
 +
<td> 1 </td>
 +
<td> 0 </td>
 +
<td> 0 </td>
 +
 +
</tr>
 +
 +
<tr>
 +
<td> 0 </td>
 +
<td> 1 </td>
 +
<td> 0 </td>
 +
 +
</tr>
 +
 +
<tr>
 +
<td> 1 </td>
 +
<td> 1 </td>
 +
<td> 1 </td>
 +
 +
</tr>
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</table> </td>
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<td> <table border="1" align="center">
 +
<col width="100">
 +
<col width="100">
 +
<col width="100">
 +
 +
<tr>
 +
<th> A</th>
 +
<th> B</th>
 +
<th> Output </th>
 +
 +
</tr>
 +
 +
<tr>
 +
<td> 0 </td>
 +
<td> 0 </td>
 +
<td> 0 </td>
 +
 +
</tr>
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 +
<tr>
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<td> 1 </td>
 +
<td> 0 </td>
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<td> 1 </td>
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</tr>
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<tr>
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<td> 0 </td>
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<td> 1 </td>
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<td> 1 </td>
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</tr>
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<tr>
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<td> 1 </td>
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<td> 1 </td>
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<td> 1 </td>
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</tr>
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</table> </td>
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</tr>
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</table>
</p>
</p>
-
<h2 class="textTitle" style="margin-top:135px;">How we make it happen?</h2>
+
<h2 class="textTitle" >How we make it happen?</h2>
<p> We accomplish the design of the switch between the two gates with the help of the site-specific recombination system.</br>
<p> We accomplish the design of the switch between the two gates with the help of the site-specific recombination system.</br>
Cre-Lox system is a typical kind of site-specific recombination system. The system consists of two components: Cre recombinase and LoxP site. It is a site-specific recombinase technology widely used to carry out excisions, insertions, translocations and inversions in the DNA of cells. It allows the DNA modification targeted to a specific cell type or be triggered by a specific external stimulus.</br>
Cre-Lox system is a typical kind of site-specific recombination system. The system consists of two components: Cre recombinase and LoxP site. It is a site-specific recombinase technology widely used to carry out excisions, insertions, translocations and inversions in the DNA of cells. It allows the DNA modification targeted to a specific cell type or be triggered by a specific external stimulus.</br>
We make use of the cre-lox site-specific system to accomplish the inversions in the DNA. When the external stimulus comes, the Cre recombinase will be expressed and then flip the sequence between the two LoxP sites.</br>
We make use of the cre-lox site-specific system to accomplish the inversions in the DNA. When the external stimulus comes, the Cre recombinase will be expressed and then flip the sequence between the two LoxP sites.</br>
-
<img src="https://static.igem.org/mediawiki/2012/9/9e/THU-APD0.png"/>
+
<img src="https://static.igem.org/mediawiki/2012/9/9e/THU-APD0.png" style="margin-left:300px;"/></br>
After the flip, the direction of the signal stream is altered, which results in changes of logic on the physical layer. Through controlling the direction of the signal stream we can achieve our goal to make a logic-flexible device. Now let’s come to a simple example.</br>
After the flip, the direction of the signal stream is altered, which results in changes of logic on the physical layer. Through controlling the direction of the signal stream we can achieve our goal to make a logic-flexible device. Now let’s come to a simple example.</br>
-
We replace the Gene of Interest with a kind of promoter. Under the initial condition, the promoter is able to induce the Output. But once the external signal comes, the expression of the Cre recombinase will be activated and it will lead to the flip between the two LoxP sites. Consequently, the promoter cannot bring out the output.
+
We replace the Gene of Interest with a kind of promoter. Under the initial condition, the promoter is able to induce the Output. But once the external signal comes, the expression of the Cre recombinase will be activated and it will lead to the flip between the two LoxP sites. Consequently, the promoter cannot bring out the output.</br>
 +
<img src="https://static.igem.org/mediawiki/2012/9/9a/THU-APD5.png"style="margin-left:150px;"/></br>
 +
 
</p>
</p>
-
<h2 class="textTitle" style="margin-top:135px;">What we exactly want and how?</h2>
+
<h2 class="textTitle" >What we exactly want and how?</h2>
-
<p> Without loss of generality, we decide to start our project with the conversion of the two basic gates mentioned above: the And gate and the Or gate. And when it comes to the situation with 2 inputs, we construct our design like this.</br>
+
<p> Without loss of generality, we decide to start our project with the conversion of the two basic gates mentioned above: the And gate and the Or gate. And when it comes to the situation with 2 inputs, we construct our design like this.</br></p>
 +
 
 +
<p style="margin-left:300px; font-size:20px;"><b>Before Reverse</b></p>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2012/e/ee/THU-APD6.png" style="margin-left:150px;"/></br></br></br>
 +
 
 +
<table border="1" align="center">
 +
<col width="100">
 +
<col width="100">
 +
<col width="100">
 +
<col width="100">
 +
<col width="100">
 +
 
 +
<tr>
 +
<th> A</th>
 +
<th> B</th>
 +
<th> Y</th>
 +
<th> X </th>
 +
<th> Output </th>
 +
 +
</tr>
 +
 
 +
<tr>
 +
<td> 0 </td>
 +
<td> 0 </td>
 +
<td> 0 </td>
 +
<td> 0 </td>
 +
<td> 0 </td>
 +
 +
</tr>
 +
 
 +
<tr>
 +
<td> 1 </td>
 +
<td> 0 </td>
 +
<td> 1 </td>
 +
<td> 0 </td>
 +
<td> 0 </td>
 +
 +
</tr>
 +
 
 +
<tr>
 +
<td> 0 </td>
 +
<td> 1 </td>
 +
<td> 0 </td>
 +
<td> 1 </td>
 +
<td> 0 </td>
 +
 +
</tr>
 +
 
 +
<tr>
 +
<td> 1 </td>
 +
<td> 1 </td>
 +
<td> 1 </td>
 +
<td> 1 </td>
 +
<td> 1 </td>
 +
 +
</tr>
 +
</table>
 +
 
 +
</br>
 +
 
 +
 
 +
<p style="margin-left:300px;font-size:20px;"><b>After Reverse</b></p>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2012/4/49/THU-APD7.png" style="margin-left:150px;"/></br>
 +
 
 +
</br>
 +
 
 +
<table border="1" align="center">
 +
<col width="100">
 +
<col width="100">
 +
<col width="100">
 +
<col width="100">
 +
<col width="100">
 +
 
 +
<tr>
 +
                <th> A</th>
 +
<th> B</th>
 +
<th> Y</th>
 +
<th> X </th>
 +
<th> Output </th>
 +
 +
</tr>
 +
 
 +
<tr>
 +
<td> 0 </td>
 +
<td> 0 </td>
 +
<td> 0 </td>
 +
<td> 0 </td>
 +
<td> 0 </td>
 +
 +
</tr>
 +
 
 +
<tr>
 +
<td> 1 </td>
 +
<td> 0 </td>
 +
<td> 1 </td>
 +
<td> 1 </td>
 +
<td> 1 </td>
 +
 +
</tr>
 +
 
 +
<tr>
 +
<td> 0 </td>
 +
<td> 1 </td>
 +
<td> 1 </td>
 +
<td> 1 </td>
 +
<td> 1 </td>
 +
 +
</tr>
 +
 
 +
<tr>
 +
<td> 1 </td>
 +
<td> 1 </td>
 +
<td> 1 </td>
 +
<td> 1 </td>
 +
<td> 1 </td>
 +
 +
</tr>
 +
</table>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<p>
 +
 
Between the LoxP site there’re promoter A&B. Initially promoter A can only induce signal X, and promoter B signal Y. The Output is under the regulation which can only be induced by X AND Y.So only when both promoter A&B are activated can we get the expression of the Output. Definitely it serves as an And gate.</br>
Between the LoxP site there’re promoter A&B. Initially promoter A can only induce signal X, and promoter B signal Y. The Output is under the regulation which can only be induced by X AND Y.So only when both promoter A&B are activated can we get the expression of the Output. Definitely it serves as an And gate.</br>
In response to the external stimulus we’ve given, the Cre recombinase come out and start the flip. After the flip either promoter A or promoter B can activate the expression of both signal X and signal Y. Then we can get the Output. So the system works as an Or gate.
In response to the external stimulus we’ve given, the Cre recombinase come out and start the flip. After the flip either promoter A or promoter B can activate the expression of both signal X and signal Y. Then we can get the Output. So the system works as an Or gate.
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</p>
</p>
 +
<h2>An Introduction to Cre-LoxP System</h2>
 +
<p>
 +
The system consists of two components: Cre recombinase and LoxP site. Cre-Lox recombination is a site-specific recombinase technology widely used to carry out deletions, insertions, translocations and inversions in the DNA of cells. It allows the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus. It is implemented both in eukaryotic and prokaryotic systems.
 +
</p>
 +
<br/>
 +
<h4 style="float:left;">I.Cre recombinase</h4>
 +
<br/><br/>
 +
<p>
 +
Cre recombinase is a 38-kDa monomeric protein encoded by bacteriophage P1. Cre binds cooperatively to loxP sites ( which remains to be elucidated later ), with one Cre monomer contacting each of two 13-bp recombinase binding elements arranged as inverted repeats around a central strand-exchange region (Fig. 1) . Cre promotes the synapsis of loxP-containing DNA substrates and catalyzes recombination between the sites, as shown in Fig. 2.
 +
</p>
 +
<img src="https://static.igem.org/mediawiki/2012/d/d6/THUAPD1.png"/>
 +
<img src="https://static.igem.org/mediawiki/2012/c/c1/THUAPD2.png"/>
 +
<h4 style="float:left;">II. LoxP site</h4>
 +
<br/><br/>
 +
<p>Lox P (locus of X-over P1) is the recognition site of Cre recombinase on the Bacteriophage P1 consisting of 34 bp. There exists an asymmetric 8 bp sequence in between with two sets of palindromic, 13 bp sequences flanking it. The detailed structure is given below.</p>
 +
<img src="https://static.igem.org/mediawiki/2012/1/16/THUAPD3.png"/>
 +
<p>
 +
The asymmetric 8bp sequence between the two palindromes is the only factor that contributes to the orientation of LoxP site. The system works in 3 ways in the presence of Cre recombinase.  Two LoxP sites with the same orientation leads to the deletion of the sequences between these two sites, while an opposite orientation leads to an inversion of the sequences between them. Two separate DNA sequences with one LoxP site respectively will integrate in the aid of Cre recombinase. The detailed mechanism will be demonstrated below.
 +
<p/>
 +
<img src="https://static.igem.org/mediawiki/2012/1/16/THUAPD3.png"/>
 +
 +
<p>
 +
A Schematic drawing of the Cre–loxP site-specific recombination pathway, based on the strand-swapping model (Nunes-Düby et al., 1995) and on Cre–loxP structural models (Guo et al., 1997;). Conserved tyrosine residues from two of the four recombinases in a synaptic tetramer cleave the DNA backbones of the recombining segments to form transient 3'-phosphotyrosine linkages. The released 5'-hydroxyl ends of the cleaved DNA undergo intermolecular nucleophilic attack of the partner phosphotyrosine linkages to complete the exchange of one pair of strands and form a Holliday intermediate. A second round of cleavages and strand exchanges using the remaining two recombinase subunits and the complementary DNA strands gives recombinant products.
 +
<p/>
 +
<h4 style="float:left;">III. Working Strategy</h4>
 +
<br/><br/>
 +
<p>
 +
The working strategy of our system is elucidated as below. The figure ( B ) depicts the sequence at work. Two promoters, which promotes transcription in opposite directions, are responsible for the transcription of genes X, X and Y ,Y respectively. After being operated upon by Cre recombinase, the sequence with the second X, promoter A and B, and the first Y  inverses and the elements mentioned above arrange in the order of X,Y,B,A,X,Y, thus A is the promoter of Y,X and B of X,Y, which altered the logic gate of the sequence. To be more precise, the statement above will be concluded schematically.
 +
<p/>
        </div>
        </div>
<a href="https://2012.igem.org/Team:Tsinghua-A/Project" style="margin-left:40px;font-size:20px;">Return</a>
<a href="https://2012.igem.org/Team:Tsinghua-A/Project" style="margin-left:40px;font-size:20px;">Return</a>

Latest revision as of 14:46, 26 October 2012

Tsinghua-A::Project::Design

What is ‘programmable‘?

Logic, which tends to be presented in the form of truth tables, shows a relationship between inputs and outputs. Below are the truth tables of the two basic gates: the And gate and the Or gate. So we quickly find the difference lies in the outputs when only one of the inputs is true. In the And gate if there are only one true input, either A or B, the output is false. But in the Or gate it’s to the contrary. So if we can control the output when only one input is true, we can successfully make the switch between the And gate and the Or gate, which can be called a programmable device.

AND Gate OR Gate
A B Output
0 0 0
1 0 0
0 1 0
1 1 1
A B Output
0 0 0
1 0 1
0 1 1
1 1 1

How we make it happen?

We accomplish the design of the switch between the two gates with the help of the site-specific recombination system.
Cre-Lox system is a typical kind of site-specific recombination system. The system consists of two components: Cre recombinase and LoxP site. It is a site-specific recombinase technology widely used to carry out excisions, insertions, translocations and inversions in the DNA of cells. It allows the DNA modification targeted to a specific cell type or be triggered by a specific external stimulus.
We make use of the cre-lox site-specific system to accomplish the inversions in the DNA. When the external stimulus comes, the Cre recombinase will be expressed and then flip the sequence between the two LoxP sites.

After the flip, the direction of the signal stream is altered, which results in changes of logic on the physical layer. Through controlling the direction of the signal stream we can achieve our goal to make a logic-flexible device. Now let’s come to a simple example.
We replace the Gene of Interest with a kind of promoter. Under the initial condition, the promoter is able to induce the Output. But once the external signal comes, the expression of the Cre recombinase will be activated and it will lead to the flip between the two LoxP sites. Consequently, the promoter cannot bring out the output.

What we exactly want and how?

Without loss of generality, we decide to start our project with the conversion of the two basic gates mentioned above: the And gate and the Or gate. And when it comes to the situation with 2 inputs, we construct our design like this.

Before Reverse




A B Y X Output
0 0 0 0 0
1 0 1 0 0
0 1 0 1 0
1 1 1 1 1

After Reverse



A B Y X Output
0 0 0 0 0
1 0 1 1 1
0 1 1 1 1
1 1 1 1 1

Between the LoxP site there’re promoter A&B. Initially promoter A can only induce signal X, and promoter B signal Y. The Output is under the regulation which can only be induced by X AND Y.So only when both promoter A&B are activated can we get the expression of the Output. Definitely it serves as an And gate.
In response to the external stimulus we’ve given, the Cre recombinase come out and start the flip. After the flip either promoter A or promoter B can activate the expression of both signal X and signal Y. Then we can get the Output. So the system works as an Or gate. That’s how we construct the switch between the And gate and the Or gate.

An Introduction to Cre-LoxP System

The system consists of two components: Cre recombinase and LoxP site. Cre-Lox recombination is a site-specific recombinase technology widely used to carry out deletions, insertions, translocations and inversions in the DNA of cells. It allows the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus. It is implemented both in eukaryotic and prokaryotic systems.


I.Cre recombinase



Cre recombinase is a 38-kDa monomeric protein encoded by bacteriophage P1. Cre binds cooperatively to loxP sites ( which remains to be elucidated later ), with one Cre monomer contacting each of two 13-bp recombinase binding elements arranged as inverted repeats around a central strand-exchange region (Fig. 1) . Cre promotes the synapsis of loxP-containing DNA substrates and catalyzes recombination between the sites, as shown in Fig. 2.

II. LoxP site



Lox P (locus of X-over P1) is the recognition site of Cre recombinase on the Bacteriophage P1 consisting of 34 bp. There exists an asymmetric 8 bp sequence in between with two sets of palindromic, 13 bp sequences flanking it. The detailed structure is given below.

The asymmetric 8bp sequence between the two palindromes is the only factor that contributes to the orientation of LoxP site. The system works in 3 ways in the presence of Cre recombinase. Two LoxP sites with the same orientation leads to the deletion of the sequences between these two sites, while an opposite orientation leads to an inversion of the sequences between them. Two separate DNA sequences with one LoxP site respectively will integrate in the aid of Cre recombinase. The detailed mechanism will be demonstrated below.

A Schematic drawing of the Cre–loxP site-specific recombination pathway, based on the strand-swapping model (Nunes-Düby et al., 1995) and on Cre–loxP structural models (Guo et al., 1997;). Conserved tyrosine residues from two of the four recombinases in a synaptic tetramer cleave the DNA backbones of the recombining segments to form transient 3'-phosphotyrosine linkages. The released 5'-hydroxyl ends of the cleaved DNA undergo intermolecular nucleophilic attack of the partner phosphotyrosine linkages to complete the exchange of one pair of strands and form a Holliday intermediate. A second round of cleavages and strand exchanges using the remaining two recombinase subunits and the complementary DNA strands gives recombinant products.

III. Working Strategy



The working strategy of our system is elucidated as below. The figure ( B ) depicts the sequence at work. Two promoters, which promotes transcription in opposite directions, are responsible for the transcription of genes X, X and Y ,Y respectively. After being operated upon by Cre recombinase, the sequence with the second X, promoter A and B, and the first Y inverses and the elements mentioned above arrange in the order of X,Y,B,A,X,Y, thus A is the promoter of Y,X and B of X,Y, which altered the logic gate of the sequence. To be more precise, the statement above will be concluded schematically.

Return