Team:ZJU-China/Design

From 2014.igem.org

(Difference between revisions)
Line 28: Line 28:
         </div>
         </div>
         <div style="display: block;" id="tab01" class="tabs">
         <div style="display: block;" id="tab01" class="tabs">
-
             <h3>1.BBa_K1433001 attB-BBa_J23110-attP</h3>
+
            <img src="https://static.igem.org/mediawiki/2014/1/1a/ZJU_support-device_04.gif" style="float:left;width:80px" />
-
             <p>BBa_J23110 is a middle strength promoter from the constitutive promoter family, which are flanked by attB and attP sites, constructing the core parts of bistable switch. The direction of the promoter, which is controlled by Int and Xis, decides the state of support device is controlled (See bistable switch page for detailed information.)int-xis-bplr.gif</p>
+
             <h3>1.BBa_K1433001 <a href="http://parts.igem.org/Part:BBa_K1433001">attB-BBa_J23110-attP</a></h3>
 +
            <img src="https://static.igem.org/mediawiki/2014/e/ee/ZJU_int-xis-bplr.gif" style="float:right;width:300px" />
 +
             <p>BBa_J23110 is a middle strength promoter from the constitutive promoter family, which are flanked by attB and attP sites, constructing the core parts of bistable switch. The direction of the promoter, which is controlled by Int and Xis, decides the state of support device is controlled (<a href="https://2014.igem.org/Team:ZJU-China/B_Switch">See bistable switch page for detailed information</a>.)</p>
         </div>
         </div>
         <div style="display: none;" id="tab02" class="tabs">
         <div style="display: none;" id="tab02" class="tabs">
 +
            <img src="https://static.igem.org/mediawiki/2014/f/f7/ZJU_support-device_05.gif" style="float:left;width:80px" />
             <h3>2. BBa_K1433005 Lambda red CDS</h3>
             <h3>2. BBa_K1433005 Lambda red CDS</h3>
-
             <p>Lambda red CDS from pKD46. See this page for its composition and function.</p>
+
             <p>Lambda red CDS from pKD46. <a href="https://2014.igem.org/Team:ZJU-China/SSR">See this page</a> for its composition and function.</p>
         </div>
         </div>
         <div style="display: none;" id="tab03" class="tabs">
         <div style="display: none;" id="tab03" class="tabs">
 +
            <img  src="https://static.igem.org/mediawiki/2014/1/11/ZJU_support-device_03.gif" style="float:left;width:80px"/>
 +
            <img  src="https://static.igem.org/mediawiki/2014/f/f7/ZJU_support-device_05.gif" style="float:left;width:80px"/>
             <h3>3. Bireporter expression device</h3>
             <h3>3. Bireporter expression device</h3>
 +
            <img src="https://static.igem.org/mediawiki/2014/5/5d/ZJU_int-flip-bp.gif" style="float:right;width:300px" />
 +
            <img src="https://static.igem.org/mediawiki/2014/c/c9/ZJU_int%2Bxis-flip_lr.gif" style="float:right;width:300px" />
 +
           
             <p>Reporter 1 &amp; reporter 2 are paired to vividly reflect the direction of promoter so as to select specific cell. Generally, there are two states in this bi-reporter expression device:
             <p>Reporter 1 &amp; reporter 2 are paired to vividly reflect the direction of promoter so as to select specific cell. Generally, there are two states in this bi-reporter expression device:
-
State 1: WORK State
+
<p>State 1: WORK State</p>
-
Lambda red &amp; reporter 1 is expressed, and support device is turned on to recombine parts.
+
<p>Lambda red &amp; reporter 1 is expressed, and support device is turned on to recombine parts.</p>
-
State 2: SELECT State
+
<p>State 2: SELECT State</p>
-
Only reporter 2 is expressed, which is used to select cells that have inserted parts.
+
<p>Only reporter 2 is expressed, which is used to select cells that have inserted parts.</p>
-
int-flip-bp.gif
+
 
-
                int+xis-flip lr.gif</p>
+
             <p>There are two kinds of combination of reporters: fluorescent proteins or antibiotic resistance genes. For the former, it is easy to get results through fluorescent tests or even by naked eyes. Besides, cells can also be measured or counted accurately by using flow cytometry, which we used in testing our bistable switch and state stability.
             <p>There are two kinds of combination of reporters: fluorescent proteins or antibiotic resistance genes. For the former, it is easy to get results through fluorescent tests or even by naked eyes. Besides, cells can also be measured or counted accurately by using flow cytometry, which we used in testing our bistable switch and state stability.
For recombination selection, resistance gene is more reliable actually because it selects candidate cells through a survival/death mode. Even the successful recombination cells are at a very low concentraion after electrotransformation, they can stilled be selected and assembled after plate cultivate.</p>
For recombination selection, resistance gene is more reliable actually because it selects candidate cells through a survival/death mode. Even the successful recombination cells are at a very low concentraion after electrotransformation, they can stilled be selected and assembled after plate cultivate.</p>
         </div>
         </div>
         <div style="display: none;" id="tab04" class="tabs">
         <div style="display: none;" id="tab04" class="tabs">
 +
            <img  src="https://static.igem.org/mediawiki/2014/e/ef/ZJU_xis_frame.png" style="float:left;width:80px"/>
             <h3>4. BBa_I0500 + BBa_1433003</h3>
             <h3>4. BBa_I0500 + BBa_1433003</h3>
-
             <p>Xis frame.png
+
             <img src="https://static.igem.org/mediawiki/2014/5/5d/ZJU_int-flip-bp.gif" style="float:right;width:300px" />
-
Xis  is controlled by pBAD promoter. When arabinose is added, Xis will be expressed, while only if Int exists will the support device turn to reporter 1 and the Lambda red system restarts.</p>
+
            <p>Xis  is controlled by pBAD promoter. When arabinose is added, Xis will be expressed, while only if Int exists will the support device turn to reporter 1 and the Lambda red system restarts.</p>
-
xis-null.gif
+
            <img src="https://static.igem.org/mediawiki/2014/6/69/ZJU_xis-null.gif" style="float:right;width:300px" />
-
             <p>AAK tag is added after Xis gene, which can accelerate the degradation rate of Xis, avoiding the interference of Int expression on the next round of attB/attP turn[1].</p>
+
             <p>AAK tag is added after Xis gene, which can accelerate the degradation rate of Xis, avoiding the interference of Int expression on the next round of attB/attP turn<sup id="ref1" name="ref1"><a href="#rf1">[1]</a></sup>.</p>
         </div>
         </div>
         <div style="display: none;" id="tab05" class="tabs">
         <div style="display: none;" id="tab05" class="tabs">
 +
            <img  src="https://static.igem.org/mediawiki/2014/4/4a/ZJU_support-device_01.gif" style="float:left;width:80px" />
 +
            <img  src="https://static.igem.org/mediawiki/2014/6/60/ZJU_support-device_02.gif" style="float:left;width:80px" />
             <h3>5. Plasmid backbone</h3>
             <h3>5. Plasmid backbone</h3>
             <p>The previous pKD46 plasmid uses a pretty good initiator oriR101 & repA101-ts (BBa_K524000) which is a low copy origin (5-10) of replication derived from the pSC101 replication origin. It is also a temperature-sensitive initiator which loses activity significantly at 37°C, totally inactivated at 42℃. Therefore, with this initiator we can easily discard the support device when circuit construction is over.</p>
             <p>The previous pKD46 plasmid uses a pretty good initiator oriR101 & repA101-ts (BBa_K524000) which is a low copy origin (5-10) of replication derived from the pSC101 replication origin. It is also a temperature-sensitive initiator which loses activity significantly at 37°C, totally inactivated at 42℃. Therefore, with this initiator we can easily discard the support device when circuit construction is over.</p>
Line 65: Line 75:
     <div class="zju_sec">
     <div class="zju_sec">
         <h3>Socket</h3>
         <h3>Socket</h3>
-
         <p>Socket is a circuit constructed on host cells, which contains homology regions and int gene. All construction happens in this circuit.(Click different parts on figure below to learn more)</p>>
+
         <p>Socket is a circuit constructed on host cells, which contains homology regions and int gene. All construction happens in this circuit.(Click different parts on figure below to learn more)</p>
         <ul class="tabs" id="tk02">
         <ul class="tabs" id="tk02">
             <li><a class="active" href="#tab11">Part 1</a></li>
             <li><a class="active" href="#tab11">Part 1</a></li>
Line 80: Line 90:
         </div>
         </div>
         <div style="display: block;" id="tab11" class="tabs">
         <div style="display: block;" id="tab11" class="tabs">
 +
            <img  src="https://static.igem.org/mediawiki/2014/1/16/ZJU_gene-socket-02.gif" style="float:left;width:80px" />
             <h3>1. Promoter BBa_J23110</h3>
             <h3>1. Promoter BBa_J23110</h3>
             <p>The promoter we use here has two functions:</p>
             <p>The promoter we use here has two functions:</p>
             <ol>
             <ol>
-
<li>To promote transcription of Int after bi-terminator unit is replaced by the inserted circuit.</li>
+
<li>To promote transcription of <em>Int</em> after bi-terminator unit is replaced by the inserted circuit.</li>
<li>To promote the inserted circuit CDS if needed.</li>
<li>To promote the inserted circuit CDS if needed.</li>
             </ol>
             </ol>
-
             <p>BBa_J23110 is a middle strength promoter from a constitutive promoter family. It can be replaced if users do not need it, but users should notice that there is a promoter behind bi-terminator unit which allows Int expression. (See solutions below)</p>
+
             <p>BBa_J23110 is a middle strength promoter from a constitutive promoter family. It can be replaced if users do not need it, but users should notice that there is a promoter behind bi-terminator unit which allows <em>Int</em> expression. (See solutions below)</p>
         </div>
         </div>
         <div style="display: none;" id="tab12" class="tabs">
         <div style="display: none;" id="tab12" class="tabs">
 +
            <img  src="https://static.igem.org/mediawiki/2014/2/23/ZJU_gene-socket-01.gif" style="float:left;width:80px" />
 +
            <img  src="https://static.igem.org/mediawiki/2014/8/8c/ZJU_gene-socket-03.gif" style="float:left;width:80px" />
             <h3>2. I-terminator unit</h3>
             <h3>2. I-terminator unit</h3>
 +
            <img src="https://static.igem.org/mediawiki/2014/6/64/ZJU_rep-Int-exp.gif" style="float:right;width:300px" />
             <p>This is the basic unit of the site of GeneSocket.</p>
             <p>This is the basic unit of the site of GeneSocket.</p>
             <p>The orange square here represents the Lambda red recombination site. Bi-terminator (BBa_B0015) is able to firmly terminate Int transcription. When the inserted part replaces the terminator, no termination exists and Int gene will be expressed.
             <p>The orange square here represents the Lambda red recombination site. Bi-terminator (BBa_B0015) is able to firmly terminate Int transcription. When the inserted part replaces the terminator, no termination exists and Int gene will be expressed.
Line 95: Line 109:
         </div>
         </div>
         <div style="display: none;" id="tab13" class="tabs">
         <div style="display: none;" id="tab13" class="tabs">
 +
            <img  src="https://static.igem.org/mediawiki/2014/4/4b/ZJU_gene-socket-04.gif" style="float:left;width:80px" />
             <h3>3. Int expression unit BBa_K1433006 BBa_K1433007 BBa_K1433008</h3>
             <h3>3. Int expression unit BBa_K1433006 BBa_K1433007 BBa_K1433008</h3>
-
             <p>Recombinase Int can flip over attB/attP sites (see bistable switch page). Besides, if Xis protein exists, Int + Xis can flip over attL/attR sites. It is crucial to tune the expression of Int to a suitable level that can reach the following balance:</p>
+
             <p>Recombinase Int can flip over attB/attP sites (<a href="https://2014.igem.org/Team:ZJU-China/B_Switch"> see bistable switch page</a>). Besides, if Xis protein exists, Int + Xis can flip over attL/attR sites. It is crucial to tune the expression of Int to a suitable level that can reach the following balance:</p>
<ol><li>Enough to flip over all attB/attP sites on the support device.</li>
<ol><li>Enough to flip over all attB/attP sites on the support device.</li>
<li>If Int cannot be over expressed otherwise there may be not enough Xis to combine with all the Int to flip over attL/attR site, causing Int+Xis and Xis function respectively, which will lead to a “Stoichiometry mismatch” (Low Xis to Int ratio can lead to bidirectional DNA inversion.)[1]</li></ol>
<li>If Int cannot be over expressed otherwise there may be not enough Xis to combine with all the Int to flip over attL/attR site, causing Int+Xis and Xis function respectively, which will lead to a “Stoichiometry mismatch” (Low Xis to Int ratio can lead to bidirectional DNA inversion.)[1]</li></ol>
Line 102: Line 117:
         <div style="display: none;" id="tab14" class="tabs">
         <div style="display: none;" id="tab14" class="tabs">
             <h3>4. The location of socket:</h3>
             <h3>4. The location of socket:</h3>
-
             <p>Our socket is inserted into bacterial chromosome in advance before any circuit construction. Therefore, at the very beginning, the socket itself is like an “insertion parts”. We use pKD46 to recombine our socket into a safe site on LacZ gene of the strain Escherichia Coli DH10B. Since LacZ is an unnecessary gene, the loss of LacZ will not have any influences on the growth of cells. Still, different locations on chromosome could have different environments [2], which further research can be down to improve. </p>
+
             <p>Our socket is inserted into bacterial chromosome in advance before any circuit construction. Therefore, at the very beginning, the socket itself is like an “insertion parts”. We use pKD46 to recombine our socket into a safe site on LacZ gene of the strain Escherichia Coli DH10B. Since LacZ is an unnecessary gene, the loss of LacZ will not have any influences on the growth of cells. Still, different locations on chromosome could have different environments <sup id="ref2" name="ref2"><a href="#rf2">[2]</a></sup>, which further research can be down to improve. </p>
         </div>
         </div>
     </div>
     </div>
Line 142: Line 157:
         </div>
         </div>
         <div style="display: block;" id="tab21" class="tabs">
         <div style="display: block;" id="tab21" class="tabs">
 +
            <img  src="https://static.igem.org/mediawiki/2014/2/2c/ZJU_Insert_part.png" style="float:left;width:80px"/>
             <h3>1. Homology region of inserted part.</h3>
             <h3>1. Homology region of inserted part.</h3>
             <p><!-- insert part.png-->
             <p><!-- insert part.png-->
Line 148: Line 164:
         </div>
         </div>
         <div style="display: none;" id="tab22" class="tabs">
         <div style="display: none;" id="tab22" class="tabs">
 +
            <img  src="https://static.igem.org/mediawiki/2014/a/ac/ZJU_standard-part_01.gif" style="float:left;width:40px" />
             <h3>2. Restriction sites</h3>
             <h3>2. Restriction sites</h3>
             <p><!--standard-part_01.gif & standard-part-pro_01.gif-->
             <p><!--standard-part_01.gif & standard-part-pro_01.gif-->
Line 155: Line 172:
         </div>
         </div>
         <div style="display: none;" id="tab23" class="tabs">
         <div style="display: none;" id="tab23" class="tabs">
 +
            <img  src="https://static.igem.org/mediawiki/2014/b/b2/ZJU_standard-part_02.gif" style="float:left;width:80px" />
             <h3>3. Bi-terminator regions</h3>
             <h3>3. Bi-terminator regions</h3>
 +
            <img src="https://static.igem.org/mediawiki/2014/2/2b/ZJU_Teminator_flipover.gif" style="float:right;width:300px" />
             <p><!--standard-part_02.gif & standard-part-pro_03.gif-->
             <p><!--standard-part_02.gif & standard-part-pro_03.gif-->
-
The bi-terminator region is inverted in standard parts. The terminator we choose here is a unidirectional terminator. It only works after Int and Xis flipping over the attL/attR site.teminator flipover.gif
+
The bi-terminator region is inverted in standard parts. The terminator we choose here is a unidirectional terminator. It only works after Int and Xis flipping over the attL/attR site.
</p>
</p>
         </div>
         </div>
         <div style="display: none;" id="tab24" class="tabs">
         <div style="display: none;" id="tab24" class="tabs">
 +
            <img  src="https://static.igem.org/mediawiki/2014/0/0b/ZJU_standard-part_03.gif" style="float:left;width:80px" />
             <h3>4. The last homology region</h3>
             <h3>4. The last homology region</h3>
             <p><!--standard-part_03.gif & standard-part-pro_04.gif-->
             <p><!--standard-part_03.gif & standard-part-pro_04.gif-->
Line 168: Line 188:
         </div>
         </div>
         <div style="display: none;" id="tab25" class="tabs">
         <div style="display: none;" id="tab25" class="tabs">
 +
            <img  src="https://static.igem.org/mediawiki/2014/8/82/ZJU_standard-part-pro_02.gif" style="float:left;width:40px" />
             <h3>5. Additional promoter</h3>
             <h3>5. Additional promoter</h3>
             <p><!--standard-part-pro_02.gif-->
             <p><!--standard-part-pro_02.gif-->
Line 179: Line 200:
         <h3>References</h3>
         <h3>References</h3>
         <hr />
         <hr />
-
         <p>[1]Bonnet, J., Subsoontorn, P. & Endy, D. Rewritable digital data storage in live cells via engineered control of recombination directionality. Proceedings of the National Academy of Sciences of the United States of America 109, 8884-8889, doi:10.1073/pnas.1202344109 (2012).</p>
+
         <p><a href="#ref1" name="rf1" id="rf1">[1]</a>Bonnet, J., Subsoontorn, P. & Endy, D. Rewritable digital data storage in live cells via engineered control of recombination directionality. Proceedings of the National Academy of Sciences of the United States of America 109, 8884-8889, doi:10.1073/pnas.1202344109 (2012).</p>
-
         <p>[2]Wargacki, A. J. et al. An engineered microbial platform for direct biofuel production from brown macroalgae. Science 335, 308-313, doi:10.1126/science.1214547 (2012).</p>
+
         <p><a href="#ref2" name="rf2" id="rf2">[2]</a>Wargacki, A. J. et al. An engineered microbial platform for direct biofuel production from brown macroalgae. Science 335, 308-313, doi:10.1126/science.1214547 (2012).</p>
     </div>
     </div>
</div>
</div>
</html>
</html>

Revision as of 20:02, 17 October 2014

Circuit design

Lack a brief intro of this page.

Support device(Click different parts on figure below to learn more )

1.BBa_K1433001 attB-BBa_J23110-attP

BBa_J23110 is a middle strength promoter from the constitutive promoter family, which are flanked by attB and attP sites, constructing the core parts of bistable switch. The direction of the promoter, which is controlled by Int and Xis, decides the state of support device is controlled (See bistable switch page for detailed information.)

Socket

Socket is a circuit constructed on host cells, which contains homology regions and int gene. All construction happens in this circuit.(Click different parts on figure below to learn more)

1. Promoter BBa_J23110

The promoter we use here has two functions:

  1. To promote transcription of Int after bi-terminator unit is replaced by the inserted circuit.
  2. To promote the inserted circuit CDS if needed.

BBa_J23110 is a middle strength promoter from a constitutive promoter family. It can be replaced if users do not need it, but users should notice that there is a promoter behind bi-terminator unit which allows Int expression. (See solutions below)

Socket.coli

Socket.coli is the final product of our project. It’s the host of GeneSocket.

In Socket.coli, the Socket circuit is inserted into bacterial chromosome in advance by using existed chromosome recombination system. The bi-terminator unit is the first site waiting for parts to be inserted. Support device is also transformed into the cell. The original state of bistable switch is WORK state, waiting for the first part to be inserted.

With this strain, GeneSocket can work as this page shows. The cell has at least 3 functions:

  1. Store GeneSocket system.
  2. Be the object of GeneSocket recombination.
  3. Easily amplify GeneSocket and spread it as a product!

Easily amplify GeneSocket and spread it as a product!

The main function of standard parts is adding the next bi-terminator unit into SOCKET together with current insertion parts, which is a crucial design for continuously circuit construction. Besides, since the circuit situation can be quite different, we provide two parts to help you deal with most cases.

1. Homology region of inserted part.

The homology regions flanking beside inserted parts are added by PCR. The Latter parts must have SpeI restriction site to link with our standard parts.

References


[1]Bonnet, J., Subsoontorn, P. & Endy, D. Rewritable digital data storage in live cells via engineered control of recombination directionality. Proceedings of the National Academy of Sciences of the United States of America 109, 8884-8889, doi:10.1073/pnas.1202344109 (2012).

[2]Wargacki, A. J. et al. An engineered microbial platform for direct biofuel production from brown macroalgae. Science 335, 308-313, doi:10.1126/science.1214547 (2012).