Team:HUST-China/Protocol

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     </li>
     </li>
     <li class="column" id="HUMANPRACTICE">
     <li class="column" id="HUMANPRACTICE">
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     <span><a href="https://2014.igem.org/Team:HUST-China/HumanPratice">Human Pratice</a></span>
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     <span><a href="https://2014.igem.org/Team:HUST-China/HumanPractice">Human Pratice</a></span>
             </li>
             </li>
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             <div class="chapter">
             <div class="chapter">
                  
                  
-
                 <span>Protocol</span>
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                 <span><font size="6px">Protocol</span></font>
                  
                  
-
                 <h1 align="left" id="h2_0"><a name="Top" id="Top"></a><a name="Part_1" id="Part_1"></a>Protocol</h1>
+
                 <h1 align="left" id="h2_0"><a name="Top" id="Top"></a><a name="Part_1" id="Part_1"></a>Part 1: The Construction of the Worker System</h1>
-
             
+
                <h3 align="left">Step 1: Gene Cloning</h3></p>
-
              <p >Our project is divided into four parts: </p>
+
                <p>To find the optimal temperature for oprF and <em>cyn</em>RTS composite amplification, we set a gradient of annealing temperature. The result shows that 58℃ is suitable to this PCR. The PCR product was stored in -20℃. The PCR reaction system was listed in table 1-1.</p>
-
<p>Part 1: The Construction of the Worker System<br />
+
                <h3 align="center">Table&nbsp;1-1 Gradient&nbsp;PCR&nbsp;System</h3>
-
  Step 1: Gene Cloning<br />
+
                <table border="1" cellpadding="1" cellspacing="0" width="800">
-
To find the optimal temperature for oprF and RTS operon amplification, we set a gradient in temperature. Then we amplified oprF and RTS sequence by PCR in 58℃. The sequence was stored in -20℃. The PCR conditions were listed as table 1-1.</p>
+
-
<p>Table&nbsp;1-1&nbsp;: Gradient&nbsp;PCR&nbsp;System</p>
+
-
<table border="1" cellpadding="1" cellspacing="0">
+
   <tr>
   <tr>
-
     <td width="123">Components(50μl)</td>
+
     <td>Components(50μl)</td>
-
     <td width="80">Volume(ml)</td>
+
     <td>Volume(ml)</td>
   </tr>
   </tr>
   <tr>
   <tr>
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   </tr>
   </tr>
   <tr>
   <tr>
-
     <td>ddH2O</td>
+
     <td>ddH<sub>2</sub>O</td>
     <td>30</td>
     <td>30</td>
   </tr>
   </tr>
</table>
</table>
-
<p>Step 2: Improvement of oprF<br />
+
<br>
-
We choose the 188th and 196th amino acid of OprF as anchor points. Designing primers (Table 2-1)to clone the target fragment, and then add the GS linker and the gene fragment coding CBP at the end of the target fragment.<br />
+
<h3 align="left">Step 2: Optimization of oprF</h3></p>
-
The protein regarding the 188th amino acid as anchor point and added GS linker was named as OprF-1 and the protein regarding the 196th amino acid as anchor point and not added GS linker was named as OprF-2.</p>
+
  <p>We choose the 188aa or 196aa of OprF as the CBP fusion points. Primers were designed (Table1-2) to clone the target fragment, and then the GS linker and/or the gene fragment coding CBP were added at the C-terminal of the target fragment.</p>
-
<p>Table&nbsp;1-2&nbsp;&nbsp;Primers&nbsp;of&nbsp;Improving&nbsp;oprF </p>
+
  <p>The protein with GS linker, using the 188aa as CBP fusion point, was named as OprF-1. While the protein without GS linker, using the 196aa as CBP fusion point, was named as OprF-2.</p>
-
<table border="1" cellpadding="1" cellspacing="0">
+
<h3 align="center">Table&nbsp;1-2&nbsp;&nbsp;Primers&nbsp;of&nbsp;Optimization&nbsp;oprF </h2>
 +
<table border="1" cellpadding="1" cellspacing="0">
   <tr>
   <tr>
     <td width="94">Primer </td>
     <td width="94">Primer </td>
-
     <td width="519">Sequence(5’→3’)</td>
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     <td width="519">Sequence(5'→3')</td>
   </tr>
   </tr>
   <tr>
   <tr>
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   </tr>
   </tr>
</table>
</table>
-
<p>&nbsp;</p>
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<h3 align="left">Step 3: Construction of Vectors</h3></p>
-
<p>Step&nbsp;3:&#160;Construction&nbsp;of&nbsp;Vectors </p>
+
<p>pET28a vector and the PCR product were digested with EcoRI and NotI from Takara, and then retrieved and purified with DNA retrieve kits from Omega.</p>
-
<p >Digest&nbsp;pET28a&nbsp;vector&nbsp;to&nbsp;donate&nbsp;a&nbsp;skeleton&nbsp;with&nbsp;EcoRI&nbsp;and&nbsp;NotI.&nbsp;Retrieve&nbsp;and&nbsp;purify&nbsp;the&nbsp;target&nbsp;genes&nbsp;with&nbsp;kits&nbsp;produced&nbsp;by&nbsp;Omega. </p>
+
<p>Then, pET28a(+) vector, gene oprF-1/oprF-2/<em>cyn</em>RTS composite were linked together with T4 ligase to form a new vector: pET28a(+)-oprF-1/oprF-2/RTS. The reaction system for digestion and conjunction were listed in the table 1-3, 1-4 and the digestion results were displayed in fig1-1.</p>
-
<p >Afterwards, pET28a(+) vector, gene oprF-1/oprF-2/RTS operon were linked together to recombine a new vector: pET28a(+)-oprF-1/oprF-2/RTS. The reaction system for digestion and conjunction were listed in the table 1-3, 1-4.</p>
+
<h3 align="center">Table 1-3  Reaction System for Digestion</h3>
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<p>Table&nbsp;1-3  &nbsp;Reaction System for Digestion</a> </p>
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<table border="1" cellpadding="1" cellspacing="0" width="800">
-
<table border="1" cellpadding="1" cellspacing="0">
+
   <tr>
   <tr>
     <td width="131">Components(50μl)</td>
     <td width="131">Components(50μl)</td>
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   </tr>
   </tr>
   <tr>
   <tr>
-
     <td>ddH2O</td>
+
     <td>ddH<sub>2</sub>O</td>
     <td>10</td>
     <td>10</td>
   </tr>
   </tr>
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   </tr>
   </tr>
</table>
</table>
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<p>Table&nbsp;1-4&nbsp;&nbsp;for&nbsp;Gene&nbsp;Conjunction</p>
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<h3 align="center">Table 1-4 for Gene Conjunction</h3>
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<table border="1" cellpadding="1" cellspacing="0">
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<table border="1" cellpadding="1" cellspacing="0" width="800px">
   <tr>
   <tr>
-
     <td width="93"><p >Components</p></td>
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     <td >Components</td>
-
     <td width="177">System components(10μl)</td>
+
     <td>System components(10μl)</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td>Solution I</td>
+
     <td>Buffer(with T4 ligase)</td>
-
     <td> 5</td>
+
     <td>5</td>
   </tr>
   </tr>
   <tr>
   <tr>
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   </tr>
   </tr>
   <tr>
   <tr>
-
     <td><p >Vector</p></td>
+
     <td>Vector</td>
     <td>0.5</td>
     <td>0.5</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td><p >Conditions</p></td>
+
     <td>Conditions</td>
-
     <td><p >16&#8451;&nbsp;1h</p></td>
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     <td>16&#8451; 1h</td>
   </tr>
   </tr>
</table>
</table>
-
<table>
+
<br>
-
<tr>
+
<h3 align="center">Fig 1-1 Digested gene and plasmid</h3>
-
<td><img src="https://static.igem.org/mediawiki/2014/d/d6/HUST_protocol_01.png" width="400" height="350"/></td>
+
<img src="https://static.igem.org/mediawiki/2014/d/d6/HUST_protocol_01.png" width="400" height="350"/></td>
-
</tr>
+
 
-
</table>
+
 
-
<p>Fig 1-1 Digested gene and plasmid</p>
+
<h3 align="left">Step 4: Transforming the Plasmids into <em>E. coli</em> BL21 Strain </h3></p>
-
<p>&nbsp;</p>
+
<p>Step&nbsp;4:&nbsp;Transforming&nbsp;the&nbsp;Plasmids&nbsp;into&nbsp;<em>E.&nbsp;coli&nbsp;</em>BL21&nbsp;Strain&nbsp; </p>
-
<p>Step&nbsp;3:&nbsp;Transferring&nbsp;the&nbsp;Plasmids&nbsp;into&nbsp;E.&nbsp;coli&nbsp;BL21&nbsp;Strain&nbsp; </p>
+
<p>Plasmids pET28a(+)-oprF-1 or pET28a(+)-oprF-2 or pET28a(+)-flA was transformed into <em>E. coli</em> BL21(DE3) and protein expression was analyzed.The strains were grown in LB medium containing 100ug/ml kanamycin at 37℃, 250rpm until OD600 reached 0.4–0.6. We then added IPTG to 0.5mM and continued the incubation at 28℃ overnight to induce the overexpression of OprF-CBP or OprF-GS-CBP or flA. The cells were collected, re-suspended with 10mM imidazole containing 0.1mM protease inhibitor PMSF and then disrupted using Selecta Sonopuls. After centrifugation, the sediment was treated with 1xSDS gel loading buffer and kept in boiling water for 5 minutes and applied to SDS-PAGE. The result was displayed in Fig 1-4.</p>
-
<p>The recombined plasmids were transferred into BL21 strain, which later was grew in LB culture medium with 100&#956;g/ml Kanamycin, 37&#8451; for one night. To ensure the conjunctions were correct, we confirmed the vectors by colonial PCR and gel electrophoresis, following the digestion of EcoRI and NotI to see the possibilities of reverse connection. The conditions for colonial PCR and dual-enzyme digestion reaction were listed in the table 1-5, 1-6. </p>
+
<h3 align="center">Table&nbsp;1-5 &nbsp;Reaction System of Colonial&nbsp;PCR validation</h3>
-
<p >Table&nbsp;1-5 &nbsp;Reaction System&nbsp;Colonial&nbsp;PCR </p>
+
<table border="1" cellpadding="1" cellspacing="0" width="800">
-
<table border="1" cellpadding="1" cellspacing="0">
+
   <tr>
   <tr>
-
     <td width="177"><p >Components(10&#956;l)</p></td>
+
     <td>Components(10&#956;l)</td>
-
     <td width="85"><p >Volume(&#109;l)</p></td>
+
     <td>Volume(&#109;l)</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td><p >2&#215;Ex&nbsp;Taq&nbsp;Mix&nbsp;</p></td>
+
     <td>2&#215;Ex&nbsp;Taq&nbsp;Mix&nbsp;</td>
     <td>5</td>
     <td>5</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td><p >Primer-F(10&#956;mol/L)&nbsp;</p></td>
+
     <td>Primer-F(10&#956;mol/L) </td>
     <td>0.3</td>
     <td>0.3</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td><p >Primer-R(10&#956;mol/L)&nbsp;</p></td>
+
     <td>Primer-R(10&#956;mol/L)&nbsp;</td>
     <td>0.3</td>
     <td>0.3</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td><p >Template</p></td>
+
     <td>Template</td>
     <td>0.3</td>
     <td>0.3</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td><p >H2O</p></td>
+
     <td>H<sub>2</sub>O</td>
     <td>4.1</td>
     <td>4.1</td>
   </tr>
   </tr>
</table>
</table>
-
<p>Table&nbsp;1-6 &nbsp;Conditions&nbsp;for&nbsp;dual-enzyme&nbsp;digestion&nbsp;reaction (37&nbsp;&#8451;&nbsp;1h)</p>
+
<h3 align="center">Table&nbsp;1-6 &nbsp;Reaction System of Double Digestion (37&nbsp;&#8451;&nbsp;1h)</h3>
<table border="1" cellpadding="1" cellspacing="0">
<table border="1" cellpadding="1" cellspacing="0">
   <tr>
   <tr>
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   </tr>
   </tr>
   <tr>
   <tr>
-
     <td><p >10&#215;H&nbsp;Buffer&nbsp;</p></td>
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     <td>10&#215;H&nbsp;Buffer&nbsp;</td>
     <td>1</td>
     <td>1</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td><p >BSA</p></td>
+
     <td>BSA</td>
     <td>1</td>
     <td>1</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td><p >EcoRI&nbsp;</p></td>
+
     <td>EcoRI&nbsp;</td>
     <td>0.5</td>
     <td>0.5</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td><p >NotI</p></td>
+
     <td>NotI</td>
     <td>0.5</td>
     <td>0.5</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td><p >ddH2O</p></td>
+
     <td>ddH<sub>2</sub>O</td>
     <td>7</td>
     <td>7</td>
-
  </tr>
 
-
  <tr>
 
-
    <td><p >Condition</a></p></td>
 
-
    <td>37 ℃ 1h</td>
 
   </tr>
   </tr>
</table>
</table>
-
<table>
+
 
-
<tr>
+
<img src="https://static.igem.org/mediawiki/2014/1/1f/HUST_protocol_02.png" width="400" height="350" /></td>
-
<td><img src="https://static.igem.org/mediawiki/2014/1/1f/HUST_protocol_02.png" width="400" height="350" /></td>
+
<h3 align="center">Fig 1-2 Gel images of novel plasmids with OprF-CBP/GS-CBP testified results</h3>
-
</tr>
+
<br>
-
</table>
+
-
<p>Fig 1-2 Gel images of novel plasmids with OprF-CBP/GS-CBP testified results</p>
+
-
<table>
+
-
<tr><td>
+
<img src="https://static.igem.org/mediawiki/2014/5/55/HUST_protocol_03.png" width="400" height="350" /></td></tr></table>
<img src="https://static.igem.org/mediawiki/2014/5/55/HUST_protocol_03.png" width="400" height="350" /></td></tr></table>
-
<p>Fig 1-3 Gel images of novel plasmids with RTS testified results</p>
+
<h3 align="center">Fig 1-3 Gel images of novel plasmids with <em>cyn</em>RTS composite testified results</h3>
-
<p>The E. coli BL21(DE3) strain transformed with pET28a(+)-flA was kindly donated by Prof. David O'Hagan and Prof. James H. Naismith from University of St Andrews, Saint Andrews, Scotland, United Kingdom.  </p>
+
<br>
-
<p>Step 4: Expression of Protein<br />
+
<p>The <em>E. coli</em> BL21(DE3) strain transformed with pET28a(+)-flA was kindly donated by Prof. David O'Hagan and Prof. James H. Naismith from University of St Andrews, Saint Andrews, Scotland, United Kingdom.  </p>
-
   Plasmid pET28a(+)-oprF-1/oprF-2/RTS/flA was transformed into E. coli BL21(DE3) and we get for protein expression analysis. The strains were grown in Luria broth containing 100ug/ml kanamycin at 37℃, 250rpm until an absorbance of 0.4–0.6 at 600 nm was reached. We then added IPTG to 0.5mM and continued the incubation at 28℃ overnight to induce the overexpression of OprF-CBP/OprF-GS-CBP. The cells were collected, suspended with 10mM imidazole containing 0.1mM protease inhibitor PMSF and then disrupted using Selecta Sonopuls. After centrifugation, the sediment was treated with 1*SDS gel loading buffer and kept in boiling water for 5 minutes and applied to SDS-PAGE. </p>
+
<h3 align="left">Step 5: Expression of Protein</h3>
-
<img src="https://static.igem.org/mediawiki/2014/c/c9/HUST_protocol_04.png"></img>
+
<p>
-
<p>Fig 1-4 Expression of Protein</p>
+
   Plasmid pET28a(+)-oprF-1/oprF-2/<em>cyn</em>RTS composite/flA was transformed into <em>E. coli</em> BL21(DE3) and we get for protein expression analysis. The strains were grown in Luria broth containing 100ug/ml kanamycin at 37℃, 250rpm until an absorbance of 0.4–0.6 at 600 nm was reached. We then added IPTG to 0.5mM and continued the incubation at 28℃ overnight to induce the overexpression of OprF-CBP/OprF-GS-CBP. The cells were collected, suspended with 10mM imidazole containing 0.1mM protease inhibitor PMSF and then disrupted using Selecta Sonopuls. After centrifugation, the sediment was treated with 1*SDS gel loading buffer and kept in boiling water for 5 minutes and applied to SDS-PAGE. </p>
-
<p>Step 5: Surface Displaying Copper Ions<br />
+
<h3 align="left">Step 6: Surface Displaying Copper Ions</h3>
-
   To identify that whether our OprF has anchored on the cell membrane of E. coli, we performed immunofluorescence assay. HA tag was added to the N-terminal of OprF-CBP so that the recombinant protein OprF-CBP-HA can be specifically recognized by anti-HA antibody. When FITC labeled anti-IgG antibody was used as the secondary antibody and interacted with the primary antibody, green fluorescence could be observed in the cell membrane of E. coli under the fluorescent microscope.</p>
+
<p>
-
<p>Part 2: The Construction of the Instructor System<br />
+
   To identify that whether our OprF has anchored on the cell membrane of <em>E. coli</em>, we performed immunofluorescence assay. HA tag was added to the N-terminal of OprF-CBP so that the recombinant protein OprF-CBP-HA can be specifically recognized by anti-HA antibody. When FITC labeled anti-IgG antibody was used as the secondary antibody and interacted with the primary antibody, green fluorescence could be observed in the cell membrane of <em>E. coli</em> under the fluorescent microscope.</p>
-
<p>All the parts except PpcoA we used to construct E. instructor are from kits in Distribution 2013.They are listed in table2-1.</p>
+
  <br>
-
<p>Table 2-1 Parts from Kits in Distribution 2013</p>
+
  <br><br>
 +
  <h1 align="left" id="h2_1"><a name="Part_2" id="Part_2"></a>Part 2: The Construction of the Instructor System</h1>
 +
  <br>
 +
  <p>All the parts except PpcoA we used to construct <em>E. instructor</em> are from kits in Distribution 2013.They are listed in table2-1.</p>
 +
<h3 align="center">Table 2-1 Parts from Kits in Distribution 2013</h3>
<table border="1" cellpadding="1" cellspacing="0">
<table border="1" cellpadding="1" cellspacing="0">
   <tr>
   <tr>
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   </tr>
   </tr>
</table>
</table>
-
<p>Step1: Plasmid construction:</p>
+
<h3>Assemble all the elements via standard restrict sites:</h3>
-
<p>Assemble all the elements via standard restrict sites:</p>
+
<h4 align="left">1. PCR: PpcoA (add EcoRI, XbaI, SpeI and PstI restriction sites)</h4>
-
<p>1. PCR: pPcoA (add EcoRI, XbaI, SpeI and PstI restriction sites)</p>
+
<img src="https://static.igem.org/mediawiki/2014/d/d3/HUST_protocol_05.png"></img>
<img src="https://static.igem.org/mediawiki/2014/d/d3/HUST_protocol_05.png"></img>
-
<p>2. Double digestion: PPcoA PCR products and rbs+CII(LVA)+ter+ter/pSB1C3(BBa_P0153):</p>
+
<br>
 +
<br>
 +
<br>
 +
<h4 align="left">2. Double digestion: PpcoA PCR products and rbs+CII(LVA)+ter+ter/pSB1C3(BBa_P0153):</h4>
<img src="https://static.igem.org/mediawiki/2014/9/95/HUST_protocol_06.png"></img>
<img src="https://static.igem.org/mediawiki/2014/9/95/HUST_protocol_06.png"></img>
-
<p>Colony PCR Validation (primer PpcoA F and PpcoA R):</p>
+
<br>
 +
<br>
 +
<br>
 +
<h4 align="left">3. Colony PCR Validation (primer PpcoA F and PpcoA R):</h4>
<img src="https://static.igem.org/mediawiki/2014/6/63/HUST_protocol_07.png"></img>
<img src="https://static.igem.org/mediawiki/2014/6/63/HUST_protocol_07.png"></img>
-
<p>The plasmid pPcoA-rbs+CII+LVA/pSB1C3 was extracted and sequenced.</p>
+
<h5>The plasmid pPcoA-rbs+CII+LVA/pSB1C3 was extracted and sequenced.</h5>
-
<p>3. Double digestion: PPcoA-rbs+CII+LVA/pSB1C3 and rbs+CI+tag-dT:</p>
+
<br>
 +
<br>
 +
<br>
 +
<h4 align="left">4. Double digestion: PpcoA-rbs+CII+LVA/pSB1C3 and rbs+CI+tag-dT:</h4>
<img src="https://static.igem.org/mediawiki/2014/f/f2/HUST_protocol_08.png"></img>
<img src="https://static.igem.org/mediawiki/2014/f/f2/HUST_protocol_08.png"></img>
-
<p>Colony PCR Validation(primer PPcoA F and cI R):</p>
+
<br>
 +
<br>
 +
<br>
 +
<h4 align="left">5. Colony PCR Validation(primer PpcoA F and cI R):</h4>
<img src="https://static.igem.org/mediawiki/2014/3/39/HUST_protocol_09.png"></img>
<img src="https://static.igem.org/mediawiki/2014/3/39/HUST_protocol_09.png"></img>
-
<p>The plasmid PPcoA-rbs+CII+LVA-rbs+CI+tag-dT/pSB1C3 was extracted and sequenced.</p>
+
<h5 align="center">The plasmid PpcoA-rbs+CII+LVA-rbs+CI+tag-dT/pSB1C3 was extracted and sequenced.</h4>
-
<p>4. Double digestion: PCR product of rbs+mRFP+tag and dT/pSB1C3:</p>
+
<br>
 +
<br>
 +
<br>
 +
<h4 align="left">6. Double digestion: PCR product of rbs+mRFP+tag and dT/pSB1C3:</h4>
<img src="https://static.igem.org/mediawiki/2014/7/70/HUST_protocol_10.png"></img>
<img src="https://static.igem.org/mediawiki/2014/7/70/HUST_protocol_10.png"></img>
-
<p>Colony PCR Validation (primer mRFP F1 and mRFP R1):</p>
+
<br>
 +
<br>
 +
<br>
 +
<h4 align="left">7. Colony PCR Validation (primer mRFP F1 and mRFP R1):</h4>
<img src="https://static.igem.org/mediawiki/2014/5/59/HUST_protocol_11.png"></img>
<img src="https://static.igem.org/mediawiki/2014/5/59/HUST_protocol_11.png"></img>
<p>The plasmid rbs+mRFP+tag-dT/pSB1C3 were extracted and sequenced.</p>
<p>The plasmid rbs+mRFP+tag-dT/pSB1C3 were extracted and sequenced.</p>
-
<p>4. Double digestion: PRE/pMD-19T and rbs+mRFP+tag-dT/pSB1C3:</p>
+
<br>
 +
<br>
 +
<br>
 +
<h4 align="left">8. Double digestion: PRE/pMD-19T and rbs+mRFP+tag-dT/pSB1C3:</h4>
<img src="https://static.igem.org/mediawiki/2014/4/4c/HUST_protocol_12.png"></img>
<img src="https://static.igem.org/mediawiki/2014/4/4c/HUST_protocol_12.png"></img>
-
<p>Monoclonal colony PCR (primer PRE F and mRFP R1):</p>
+
<br>
 +
<h4 align="left">9. Colony PCR Validation (primer PRE F and mRFP R1):</h4>
<img src="https://static.igem.org/mediawiki/2014/f/f6/HUST_protocol_13.png"></img>
<img src="https://static.igem.org/mediawiki/2014/f/f6/HUST_protocol_13.png"></img>
-
<p>The plasmid PRE- rbs+mRFP+tag-dT/pMD19-T was extracted and sequenced.</p>
+
<p h5 align="left">The plasmid PRE-rbs+mRFP+tag-dT/pMD19-T was extracted and sequenced.</h4></p>
-
<p>Double digestion:</p>
+
<br>
-
<p>Step2: Transformation of bacteria and colony PCR(primer VF2 and VR):</p>
+
<br>
-
<p>Promoter lambda (cI regulated) with GFP (+LVA) reporter in pSB1A3:</p>
+
<p><h3>step 2: Transformation of bacteria and colony PCR(primer VF2 and VR)</h3></p>
 +
<h4 align="left">1. Promoter lambda (cI regulated) with GFP (+LVA) reporter in pSB1A3:</h4>
<img src="https://static.igem.org/mediawiki/2014/5/59/HUST_protocol_14.png"></img>
<img src="https://static.igem.org/mediawiki/2014/5/59/HUST_protocol_14.png"></img>
-
<p>The plasmid PR-rbs+GFP+LVA/pSB1C3 was extracted and sequenced.</p>
+
<h4 align="left">The plasmid PR-rbs+GFP+LVA/pSB1C3 was extracted and sequenced.</h4>
-
<p>Double digestion:(3A assembly)</p>
+
<br>
-
<p>PRE- rbs+mRFP+tag-dT/pMD19-T</p>
+
<h4 align="left">2. Double digestion:(3A assembly)</h4>
 +
<h4 align="left"> PRE- rbs+mRFP+tag-dT/pMD19-T</h4>
<img src="https://static.igem.org/mediawiki/2014/8/88/HUST_protocol_15.png"></img>
<img src="https://static.igem.org/mediawiki/2014/8/88/HUST_protocol_15.png"></img>
-
<p>PPcoA-rbs+CII+LVA-rbs+CI+tag-dT/pSB1C3</p>
+
<br>
-
<img src="https://static.igem.org/mediawiki/2014/8/84/HUST_protocol_16.png"></img>
+
<br>
-
<p>Colony PCR validation (primer cI F and mRFP R1):</p>
+
<br>
 +
<h4 align="left">3. PpcoA-rbs+CII+LVA-rbs+CI+tag-dT/pSB1C3</p>
 +
<p style="align:left"><img src="https://static.igem.org/mediawiki/2014/8/84/HUST_protocol_16.png"></img></p>
 +
<br>
 +
<br>
 +
<br>
 +
<h3 align="left">4. Colony PCR validation (primer cI F and mRFP R1):</h4>
<img src="https://static.igem.org/mediawiki/2014/6/6f/HUST_protocol_17.png"></img>
<img src="https://static.igem.org/mediawiki/2014/6/6f/HUST_protocol_17.png"></img>
-
<p>The plasmid PPcoA-rbs+CII+LVA-rbs+CI+tag-dT-PRE-rbs+mRFP+tag-dT/pSB1C3 was extracted and sequenced using primer VF2 and VR.</p>
+
<h4 align="left">The plasmid PpcoA-rbs+CII+LVA-rbs+CI+tag-dT-PRE-rbs+mRFP+tag-dT/pSB1C3 was extracted and sequenced using primer VF2 and VR.</h4>
-
<p>Double digestion: </p>
+
<h3>5. Double digestion PR-rbs+GFP+LVA/pSB1C3</h3>
-
<p>PR-rbs+GFP+LVA/pSB1C3</p>
+
<img src="https://static.igem.org/mediawiki/2014/7/7b/HUST_protocol_18.png"></img>
<img src="https://static.igem.org/mediawiki/2014/7/7b/HUST_protocol_18.png"></img>
-
<p>PPcoA-rbs+CII+LVA-rbs+CI+tag-dT-PRE-rbs+mRFP+tag-dT/pSB1C3</p>
+
<br>
 +
<br>
 +
<br>
 +
<h4 align="left">6. PpcoA-rbs+CII+LVA-rbs+CI+tag-dT-PRE-rbs+mRFP+tag-dT/pSB1C3</h4>
<img src="https://static.igem.org/mediawiki/2014/1/10/HUST_protocol_19.png"></img>
<img src="https://static.igem.org/mediawiki/2014/1/10/HUST_protocol_19.png"></img>
-
<p>Monoclonal colony PCR (primer PRE F and GFP R):</p>
+
<br>
 +
<h4 align="left">7. Colony PCR Validation (primer PRE F and GFP R):</h4>
<img src="https://static.igem.org/mediawiki/2014/c/ce/HUST_protocol_20.png"></img>
<img src="https://static.igem.org/mediawiki/2014/c/ce/HUST_protocol_20.png"></img>
-
<p>The plasmid PPcoA-rbs+CII+LVA-rbs+CI+tag-dT-PRE-rbs+mRFP+tag-dT-PR-rbs+GFP+LVA/pSB1C3 was extracted and sequenced using primer VF2 and VR.</p>
+
<p><h4 align="left">The plasmid PpcoA-rbs+CII+LVA-rbs+CI+tag-dT-PRE-rbs+mRFP+tag-dT-PR-rbs+GFP+LVA/pSB1C3 was extracted and sequenced using primer VF2 and VR.</h4></p>
-
<p>Part 3: The Construction of the Kill Switch</br>
+
<br>
-
<p>Plasmid construction:</p>
+
<br>
-
<p>PCR: CI repressor from E. coli phage lambda (+LVA) in pSB1C3(BBa_C0051)was amplified to add RBS using primer:</p>
+
<br>
-
<p>cI F: 5' –TATGAATTCTCTAGATAAGGAGATATAATGAGCACAAAAAAG-3'</p>
+
 
-
<p>and cI R: 5' –TAATCTGCAGACTAGTGCGATCTACACTAGCACTATC-3'</p>
+
 
 +
<h1 align="left" id="h2_1"><a name="Part_3" id="Part_3"></a>Part 3: The Construction of the Kill Switch</h1>
 +
<h3>Plasmid construction:</h3>
 +
<p>PCR: CI repressor from <em>E. coli</em> phage lambda (+LVA) in pSB1C3(BBa_C0051)was amplified to add RBS using primer:</p>
 +
<p>cI F: 5'–TATGAATTCTCTAGATAAGGAGATATAATGAGCACAAAAAAG-3' and cI R: 5'–TAATCTGCAGACTAGTGCGATCTACACTAGCACTATC-3'</p>
<img src="https://static.igem.org/mediawiki/2014/4/48/HUST_protocol_20_5.png"></img>
<img src="https://static.igem.org/mediawiki/2014/4/48/HUST_protocol_20_5.png"></img>
-
<p>Double digestion: PCR product of cI+rbs obtained using gel extraction kit and double terminator (dT) in pSB1C3 were applied to double digestion. The reaction systems were listed in table 3-1, 3-2.</p>
+
<br>
-
<p>Table3-1 Digestion of Fragment (cI+rbs):</p>
+
<h3>Double digestion:<h3><p> PCR product of cI+rbs obtained using gel extraction kit and double terminator (dT) in pSB1C3 were applied to double digestion. The reaction systems were listed in table 3-1, 3-2.</p>
 +
<h3 align="center">Table3-1 Digestion of Fragment (cI+rbs):</h3>
<table border="1" cellpadding="1" cellspacing="0">
<table border="1" cellpadding="1" cellspacing="0">
   <tr>
   <tr>
Line 469: Line 507:
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td>EcoR I</td>
+
     <td>EcoRI</td>
     <td>2μl</td>
     <td>2μl</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td>Spe I</td>
+
     <td>SpeI</td>
     <td>2 μl</td>
     <td>2 μl</td>
   </tr>
   </tr>
Line 481: Line 519:
   </tr>
   </tr>
</table>
</table>
-
<p>Table 3-2 Digestion of pSB1C3-dT:</p>
+
<br>
 +
<h3 align="center">Table 3-2 Digestion of pSB1C3-dT:</h3>
<table border="1" cellpadding="1" cellspacing="0">
<table border="1" cellpadding="1" cellspacing="0">
   <tr>
   <tr>
Line 492: Line 531:
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td>EcoR I</td>
+
     <td>EcoRI</td>
     <td>2μl</td>
     <td>2μl</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td>Spe I</td>
+
     <td>SpeI</td>
     <td>2 μl</td>
     <td>2 μl</td>
   </tr>
   </tr>
Line 507: Line 546:
<img src="https://static.igem.org/mediawiki/2014/9/9e/HUST_protocol_21.png"></img>
<img src="https://static.igem.org/mediawiki/2014/9/9e/HUST_protocol_21.png"></img>
<p>Then, we linked them together at room temperature for 1h. The reaction system listed in table 3-3. </p>
<p>Then, we linked them together at room temperature for 1h. The reaction system listed in table 3-3. </p>
-
<p>Table 3-3 Reaction System of  Ligation</p>
+
<h3 align="center">Table 3-3 Reaction System of  Ligation</h3>
<table border="1" cellpadding="1" cellspacing="0">
<table border="1" cellpadding="1" cellspacing="0">
   <tr>
   <tr>
Line 526: Line 565:
   </tr>
   </tr>
</table>
</table>
-
<p>Colony PCR validation:</p>
+
<h3>Colony PCR validation:</h3>
<p>Select monoclonal and applied to PCR:</p>
<p>Select monoclonal and applied to PCR:</p>
<img src="https://static.igem.org/mediawiki/2014/2/2a/HUST_protocol_22.png"></img>
<img src="https://static.igem.org/mediawiki/2014/2/2a/HUST_protocol_22.png"></img>
<p>The positive colone was cultured in LB overnight for plasmid extraction and sequencing.</p>
<p>The positive colone was cultured in LB overnight for plasmid extraction and sequencing.</p>
 +
<br>
<p>Assemble all elements one by one using the same method:</p>
<p>Assemble all elements one by one using the same method:</p>
<p>Double digestion: </p>
<p>Double digestion: </p>
-
<p>P<sub>L</sub> lacI (BBa_R0011) in pSB1A3 and pSB1C3-cI+rbs-dT</p>
+
<h3 >P<sub>L</sub> lacI (BBa_R0011) in pSB1A3 and pSB1C3-cI+rbs-dT</h3>
<img src="https://static.igem.org/mediawiki/2014/4/46/HUST_protocol_23.png"></img>
<img src="https://static.igem.org/mediawiki/2014/4/46/HUST_protocol_23.png"></img>
-
<p>Colony PCR validation(primer VF2 and cI R):</p>
+
<br>
 +
<h3>Colony PCR validation(primer VF2 and cI R):</h3>
<img src="https://static.igem.org/mediawiki/2014/5/55/HUST_protocol_24.png"></img>
<img src="https://static.igem.org/mediawiki/2014/5/55/HUST_protocol_24.png"></img>
-
<p>The plasmid PL lacI-rbs+cI-dT/pSB1C3 was extracted and sequenced.</p>
+
<br>
-
<p>Double digestion:</p>
+
<h3>The plasmid PL lacI-rbs+cI-dT/pSB1C3 was extracted and sequenced.</h3>
-
<p>P<sub>L</sub>/pMD-19T and rbs+mRFP+tag PCR products:</p>
+
<h3>Double digestion:</h3>
 +
<h3>P<sub>L</sub>/pMD-19T and rbs+mRFP+tag PCR products:</h3>
<img src="https://static.igem.org/mediawiki/2014/1/12/HUST_protocol_25.png"></img>
<img src="https://static.igem.org/mediawiki/2014/1/12/HUST_protocol_25.png"></img>
<p>Colony PCR validation(primer VF2 and mRFP R1):</p>
<p>Colony PCR validation(primer VF2 and mRFP R1):</p>
<img src="https://static.igem.org/mediawiki/2014/1/14/HUST_protocol_26.png"></img>
<img src="https://static.igem.org/mediawiki/2014/1/14/HUST_protocol_26.png"></img>
-
<p>Colony PCR validation(primer VF2 and mRFP R1):</p>
+
<br>
 +
<h3>Colony PCR validation(primer VF2 and mRFP R1):</h3>
-
<p>The plasmid PL -rbs+mRFP+tag/pSB1C3 was extracted and sequenced.</p>
+
<h3>The plasmid PL -rbs+mRFP+tag/pSB1C3 was extracted and sequenced.</h3>
-
<p>Double digestion:</p>
+
<h3>Double digestion:</p>
-
<p>P<sub>L</sub> -rbs+mRFP+tag/pMD19-T and PL lacI-rbs+cI-dT/pSB1C3:</p>
+
<h3>P<sub>L</sub> -rbs+mRFP+tag/pMD19-T and PL lacI-rbs+cI-dT/pSB1C3:</h3>
<img src="https://static.igem.org/mediawiki/2014/4/45/HUST_protocol_27.png"></img>
<img src="https://static.igem.org/mediawiki/2014/4/45/HUST_protocol_27.png"></img>
-
<p>Colony PCR validation (primer VF2 and cI R):</p>
+
<br>
 +
<h3>Colony PCR validation (primer VF2 and cI R):</h3>
<img src="https://static.igem.org/mediawiki/2014/8/8d/HUST_protocol_28.png"></img>
<img src="https://static.igem.org/mediawiki/2014/8/8d/HUST_protocol_28.png"></img>
<p>The plasmid P<sub>L</sub> lacI-rbs+cI-dT- PL -rbs+mRFP+tag/pMD-19T was extracted and sequenced.</p>
<p>The plasmid P<sub>L</sub> lacI-rbs+cI-dT- PL -rbs+mRFP+tag/pMD-19T was extracted and sequenced.</p>
-
<p>Part 4: Measuring of Growth Curve</p>
+
<br><br>
-
<p>Step 1: 200μL overnight-cultured bacterial was added to vials containing newly prepared culture medium (containing 100μg/ml Kanamycin). Set one vial with culture medium as blank. All the samples were cultured in the shaking incubator with rotational speed at 200 rpm and temperature at 37℃.</p>
+
<br>
 +
<h1 align="left" id="h2_1"><a name="Part_4" id="Part_4"></a>Part 4: Measuring of Growth Curve</h1>
-
<p>Step 2: OD600 values was measured using the nucleic acid analyzer. When the OD600 values of culture reached 0.6, 200μL culture was added to a new vial containing 5mL LB, 100μg/ml Kanamycin, 0.1M IPTG and CN-/F-/Cu2+ with different concentration. Set one vial without bacteria as blank. All these samples were cultured in the shaking incubator with rotational speed at 200 rpm and temperature at 37℃.</p>
+
<h3 align="left">Step 1:</h2><p> 200μL overnight-cultured bacterial was added to vials containing newly prepared culture medium (containing 100μg/ml Kanamycin). Set one vial with culture medium as blank. All the samples were cultured in the shaking incubator with rotational speed at 200 rpm and temperature at 37℃.</p>
-
<p>Step 3: OD600 of samples from the vials were measured every 30 min. </p>
+
<h3 align="left">Step 2:</h2><p> OD<sub>600</sub> values was measured using the nucleic acid analyzer. When the OD<sub>600</sub> values of culture reached 0.6, 200μL culture was added to a new vial containing 5mL LB, 100μg/ml Kanamycin, 0.1M IPTG and CN-/F-/Cu2+ with different concentration. Set one vial without bacteria as blank. All these samples were cultured in the shaking incubator with rotational speed at 200 rpm and temperature at 37℃.</p>
-
<p>Part 5: The Standardization of Parts</p>
+
<h3 align="left">Step 3:</h2> <p> OD600 of samples from the vials were measured every 30 min. </p>
-
<p>Materials (used in this part)</p>
+
<br><br>
-
<p>The materials we used in this part are listed in table 5-1 and table 5-2</p>
+
<br>
-
<p>Table 5-1: Bacterial strains and plasmids</p>
+
<p h1 align="left" id="h2_1"><a name="Part_5" id="Part_5"></a>Part 5: The Standardization of Parts</h1></p>
 +
<h2>Materials (used in this part)</h2>
 +
<h4>The materials we used in this part are listed in table 5-1 and table 5-2</h3>
 +
<h3 align="center">Table 5-1: Bacterial strains and plasmids</h3>
<table border="1" cellpadding="1" cellspacing="0">
<table border="1" cellpadding="1" cellspacing="0">
   <tr>
   <tr>
Line 569: Line 617:
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td>E. coli DH5α</td>
+
     <td><em>E. coli</em> DH5α</td>
     <td>Strains</td>
     <td>Strains</td>
     <td>conserved in the lab<td>
     <td>conserved in the lab<td>
Line 584: Line 632:
   </tr>
   </tr>
</table>
</table>
-
<p>Table 5-2: Primers' Name and Sequence</p>
+
<br>
 +
<h3 align="center">Table 5-2: Primers' Name and Sequence</h3>
<table border="1" cellpadding="1" cellspacing="0">
<table border="1" cellpadding="1" cellspacing="0">
   <tr>
   <tr>
Line 607: Line 656:
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td>RTS operon-F</td>
+
     <td>RTS-F</td>
     <td>G GAATTC GCGGCCGC T TCTAGA G TCAGAACGGTTTGG</td>
     <td>G GAATTC GCGGCCGC T TCTAGA G TCAGAACGGTTTGG</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td>RTS operon-R</td>
+
     <td>RTS-R</td>
     <td>TGCA CTGCAG CGGCCGC T ACTAGT ACTACCGTGATTCATTTC</td>
     <td>TGCA CTGCAG CGGCCGC T ACTAGT ACTACCGTGATTCATTTC</td>
   </tr>
   </tr>
Line 655: Line 704:
   </tr>
   </tr>
</table>
</table>
-
<p>Step 1: Gene Standardized</p>
+
<br>
-
<p>PCR amplification with the primers set as the template (pET28a-OprF-CBP/OprF-GS-CBP/RTS/FLA). The conditions of the reaction were listed in table 5-3. </p>
+
<h3 align="left">Step 1: Gene Standardized</h2>
-
<p>Table 5-3: The Reaction System to harvest target genes</p>
+
<h3>P4R amplification with the primers set as the template (pET28a-OprF-CBP/OprF-GS-CBP/<em>cyn</em>RTS composite/FLA). The conditions of the reaction were listed in table 5-3. </h3>
 +
<h3 align="center">Table 5-3: The Reaction System to harvest target genes</h3>
<table border="1" cellpadding="1" cellspacing="0">
<table border="1" cellpadding="1" cellspacing="0">
<tr>  
<tr>  
Line 688: Line 738:
</tr>
</tr>
<tr>
<tr>
-
   <td>ddH2O </td>
+
   <td>ddH<sub>2</sub>O </td>
   <td>35.75</td>
   <td>35.75</td>
</tr>
</tr>
</table>
</table>
-
<p>Step 2: Obliteration of the Illegal Restriction Sites in the Gene</p>
+
<br>
 +
<h3 align="left">Step 2: Obliteration of the Illegal Restriction Sites in the Gene</h2>
<p>It is necessary to add standard restriction enzyme sites-EcoRI/XbaI/SpeI/PstI to both terminals of each gene. However, the sequence of ompC contains EcoRI, SpeI and PstI restriction site. So we obliterated the restriction sites by site-directed mutagenesis based on overlap extension PCR. Briefly, target mutation (GAATTC→GAGTTC, ACTAGT→GCTAGT , CTGCAG→CTCCAA, ) was introduced into primers (ompC-m-A/B/C/D/E/F/G/H in table 5-2), and the four previous PCR products(ompC-AB, ompC-CD, ompC-EF, ompC-GH) were used as template for the second PCR, and the two previous PCR products(ompC-AD, ompC-EH) were used as template for the third PCR. The final PCR segment with target mutation sites was then cloned into pMD18-T vector for sequencing. The three PCR systems were listed in table 5-4, 5-5, 5-6. </p>
<p>It is necessary to add standard restriction enzyme sites-EcoRI/XbaI/SpeI/PstI to both terminals of each gene. However, the sequence of ompC contains EcoRI, SpeI and PstI restriction site. So we obliterated the restriction sites by site-directed mutagenesis based on overlap extension PCR. Briefly, target mutation (GAATTC→GAGTTC, ACTAGT→GCTAGT , CTGCAG→CTCCAA, ) was introduced into primers (ompC-m-A/B/C/D/E/F/G/H in table 5-2), and the four previous PCR products(ompC-AB, ompC-CD, ompC-EF, ompC-GH) were used as template for the second PCR, and the two previous PCR products(ompC-AD, ompC-EH) were used as template for the third PCR. The final PCR segment with target mutation sites was then cloned into pMD18-T vector for sequencing. The three PCR systems were listed in table 5-4, 5-5, 5-6. </p>
-
<p>Table 5-4: Reaction System of the 1st PCR</p>
+
<h3 align="center">Table 5-4: Reaction System of the 1st PCR</h3>
<table border="1" cellpadding="1" cellspacing="0">
<table border="1" cellpadding="1" cellspacing="0">
<tr>  
<tr>  
Line 717: Line 768:
</tr>
</tr>
<tr>
<tr>
-
   <td>H2O </td>
+
   <td>H<sub>2</sub>O </td>
   <td>20.5</td>
   <td>20.5</td>
</tr>
</tr>
</table>
</table>
-
<p>Table 5-5: Reaction System of the 2nd PCR</p>
+
<br>
 +
<h3 align="center">Table 5-5: Reaction System of the 2nd PCR</h3>
<table border="1" cellpadding="1" cellspacing="0">
<table border="1" cellpadding="1" cellspacing="0">
<tr>  
<tr>  
Line 752: Line 804:
</tr>
</tr>
</table>
</table>
-
<p>Table 5-6: Reaction System of 3rd PCR</p>
+
<br>
 +
<h3 align="center">Table 5-6: Reaction System of 3rd PCR</h3>
<table border="1" cellpadding="1" cellspacing="0">
<table border="1" cellpadding="1" cellspacing="0">
<tr>  
<tr>  
Line 779: Line 832:
</tr>
</tr>
<tr>
<tr>
-
   <td>H2O </td>
+
   <td>H<sub>2</sub>O </td>
   <td>10</td>
   <td>10</td>
</tr>
</tr>
</table>
</table>
-
<p>Step 3: Insertion of the Standard Parts into plasmid backbone-pSB1C3</p>
+
<br>
 +
<h3 align="left">Step 3: Insertion of the Standard Parts into plasmid backbone-pSB1C3</h2>
<p>pSB1C3 backbone and the standardized parts was digested with EcoRI and PstI and then retrieved and purified with kits from Omega. pSB1C3 and the standardized parts were then linked together to from new Biobricks.</p>
<p>pSB1C3 backbone and the standardized parts was digested with EcoRI and PstI and then retrieved and purified with kits from Omega. pSB1C3 and the standardized parts were then linked together to from new Biobricks.</p>
-
<p>Step 4 : Submission of New Standardized Parts</p>
+
<br>
 +
<h3 align="left">Step 4 : Submission of New Standardized Parts</h2>
<p>After completing all the validation, the four parts were submitted to iGEM official organization in the early October and arrived in New York in 8th Oct. The information was listed in the table 5-7.</p>
<p>After completing all the validation, the four parts were submitted to iGEM official organization in the early October and arrived in New York in 8th Oct. The information was listed in the table 5-7.</p>
<table border="1" cellpadding="1" cellspacing="0">
<table border="1" cellpadding="1" cellspacing="0">
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   <td>pSB1C3-oprF-1(EcoRI PstI)</td>
   <td>pSB1C3-oprF-1(EcoRI PstI)</td>
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   <td>A major outer membrane protein of Pseudomonas aeruginosa functions as a nonspecific porin to allow the passage of small hydrophilic molecules. We cut the protein at Val188 and add a CBP tag to C-terminal with a GS linker between them.</td>
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   <td>A major outer membrane protein of <em>Pseudomonas aeruginosa</em> functions as a nonspecific porin to allow the passage of small hydrophilic molecules. We cut the protein at 188aa and add a CBP tag to C-terminal with a GS linker between them.</td>
</tr>
</tr>
<tr>
<tr>
   <td>pSB1C3-oprF-2(EcoRI PstI)</td>
   <td>pSB1C3-oprF-2(EcoRI PstI)</td>
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   <td>A major outer membrane protein of Pseudomonas aeruginosa functions as a nonspecific porin to allow the passage of small hydrophilic molecules. We cut the protein at Ala196 and add a CBP tag to C-terminal.</td>
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   <td>A major outer membrane protein of <em>Pseudomonas aeruginosa<em> functions as a nonspecific porin to allow the passage of small hydrophilic molecules. We cut the protein at 196aa and add a CBP tag to C-terminal.</td>
</tr>
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<tr>
<tr>
   <td>pSB1C3-ompC(EcoRI PstI)</td>
   <td>pSB1C3-ompC(EcoRI PstI)</td>
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   <td>Outer membrane porin protein C from Escherichia coli BL21(DE3)</td>
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   <td>Outer membrane porin protein C from <em>Escherichia coli</em> BL21(DE3)</td>
</tr>
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<tr>
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   <td>pSB1C3-RTS(EcoRI PstI)</td>
   <td>pSB1C3-RTS(EcoRI PstI)</td>
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   <td>Cyn operon in Escherichia coli BL21(DE3) (cynR+cynT+cynS) is about cyanate detoxification.</td>
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   <td>Cyn operon in <em>Escherichia coli</em> BL21(DE3) (cynR+cynT+cynS) is about cyanate detoxification.</td>
</tr>
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<tr>
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             <div class="anchor-h2" id="h2num_1"><p class="h2_0"><a href="#Part_1">Part 1</a></p></div>
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             <div class="anchor-h2" id="h2num_1"><p class="h2_1"><a href="#Part_2">Part 2</a></p></div>
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             <div class="anchor-h2" id="h2num_1"><p style="text-align:right"class="h2_0"><a href="#Part_1">Worker System</a></p></div>
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             <div class="anchor-h2" id="h2num_1"><p class="h2_2"><a href="#Part_3">Part 3</a></p></div>
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             <div class="anchor-h2" id="h2num_1"><p style="text-align:right"class="h2_1"><a href="#Part_2">Instructor System</a></p></div>
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             <div class="anchor-h2" id="h2num_1"><p class="h2_3"><a href="#References">References</a></p></div>
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             <div class="anchor-h2" id="h2num_1"><p style="text-align:right"class="h2_2"><a href="#Part_3">Kill Switch</a></p></div>
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             <div class="anchor-h2" id="h2num_1"><p style="text-align:right"class="h2_2"><a href="#Part_4">Growth Curve</a></p></div>
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            <div class="anchor-h2" id="h2num_1"><p style="text-align:right"class="h2_2"><a href="#Part_5">Standardization</a></p></div>
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Latest revision as of 01:51, 18 October 2014

oo

Protocol

Part 1: The Construction of the Worker System

Step 1: Gene Cloning

To find the optimal temperature for oprF and cynRTS composite amplification, we set a gradient of annealing temperature. The result shows that 58℃ is suitable to this PCR. The PCR product was stored in -20℃. The PCR reaction system was listed in table 1-1.

Table 1-1 Gradient PCR System

Components(50μl) Volume(ml)
PrimerStar Buffer 10
dNTPs(2.5mM) 5
Primer-F(10μM) 1.5
Primer-R(10μM) 1.5
Template 1.5
PrimerStar 0.5
ddH2O 30

Step 2: Optimization of oprF

We choose the 188aa or 196aa of OprF as the CBP fusion points. Primers were designed (Table1-2) to clone the target fragment, and then the GS linker and/or the gene fragment coding CBP were added at the C-terminal of the target fragment.

The protein with GS linker, using the 188aa as CBP fusion point, was named as OprF-1. While the protein without GS linker, using the 196aa as CBP fusion point, was named as OprF-2.

Table 1-2  Primers of Optimization oprF

Primer Sequence(5'→3')
oprF-CBP-F CCGGAATTCAACTGAAGAACACCTT
oprF-CBP-R1 CCAGCCGCCATGATGCGGGGAAACCGGTTCCGGAGCCGGAGCGGC
oprF-CBP-R2 ATAGTTTAGCGGCCGCCGGCCAGCCGCCATGATGCGGGGA
oprF-GS-F CCGGAATTCAACTGAAGAACACCTT
oprF-GS-R1 TGAACCTCCGCCACCTTTCGAACCACCGAAGTTGAAG
oprF-GS-R2 CCAGCCGCCATGATGCGGGGATGAACCTCCGCCACC
oprF-GS-R3 ATAGTTTAGCGGCCGCCGGCCAGCCGCCATGATGCGGGGA

Step 3: Construction of Vectors

pET28a vector and the PCR product were digested with EcoRI and NotI from Takara, and then retrieved and purified with DNA retrieve kits from Omega.

Then, pET28a(+) vector, gene oprF-1/oprF-2/cynRTS composite were linked together with T4 ligase to form a new vector: pET28a(+)-oprF-1/oprF-2/RTS. The reaction system for digestion and conjunction were listed in the table 1-3, 1-4 and the digestion results were displayed in fig1-1.

Table 1-3 Reaction System for Digestion

Components(50μl) Volume(μl)
10×H Buffer 5
BSA 5
EcoRI 2.5
NotI 2.5
Fragment 25
ddH2O 10
Conditions 37℃ 1h

Table 1-4 for Gene Conjunction

Components System components(10μl)
Buffer(with T4 ligase) 5
Insert 4.5
Vector 0.5
Conditions 16℃ 1h

Fig 1-1 Digested gene and plasmid

Step 4: Transforming the Plasmids into E. coli BL21 Strain

Step 4: Transforming the Plasmids into E. coli BL21 Strain 

Plasmids pET28a(+)-oprF-1 or pET28a(+)-oprF-2 or pET28a(+)-flA was transformed into E. coli BL21(DE3) and protein expression was analyzed.The strains were grown in LB medium containing 100ug/ml kanamycin at 37℃, 250rpm until OD600 reached 0.4–0.6. We then added IPTG to 0.5mM and continued the incubation at 28℃ overnight to induce the overexpression of OprF-CBP or OprF-GS-CBP or flA. The cells were collected, re-suspended with 10mM imidazole containing 0.1mM protease inhibitor PMSF and then disrupted using Selecta Sonopuls. After centrifugation, the sediment was treated with 1xSDS gel loading buffer and kept in boiling water for 5 minutes and applied to SDS-PAGE. The result was displayed in Fig 1-4.

Table 1-5  Reaction System of Colonial PCR validation

Components(10μl) Volume(ml)
2×Ex Taq Mix  5
Primer-F(10μmol/L) 0.3
Primer-R(10μmol/L)  0.3
Template 0.3
H2O 4.1

Table 1-6  Reaction System of Double Digestion (37 ℃ 1h)

Components(10μl) Volume(μl)
10×H Buffer  1
BSA 1
EcoRI  0.5
NotI 0.5
ddH2O 7

Fig 1-2 Gel images of novel plasmids with OprF-CBP/GS-CBP testified results


Fig 1-3 Gel images of novel plasmids with cynRTS composite testified results


The E. coli BL21(DE3) strain transformed with pET28a(+)-flA was kindly donated by Prof. David O'Hagan and Prof. James H. Naismith from University of St Andrews, Saint Andrews, Scotland, United Kingdom.

Step 5: Expression of Protein

Plasmid pET28a(+)-oprF-1/oprF-2/cynRTS composite/flA was transformed into E. coli BL21(DE3) and we get for protein expression analysis. The strains were grown in Luria broth containing 100ug/ml kanamycin at 37℃, 250rpm until an absorbance of 0.4–0.6 at 600 nm was reached. We then added IPTG to 0.5mM and continued the incubation at 28℃ overnight to induce the overexpression of OprF-CBP/OprF-GS-CBP. The cells were collected, suspended with 10mM imidazole containing 0.1mM protease inhibitor PMSF and then disrupted using Selecta Sonopuls. After centrifugation, the sediment was treated with 1*SDS gel loading buffer and kept in boiling water for 5 minutes and applied to SDS-PAGE.

Step 6: Surface Displaying Copper Ions

To identify that whether our OprF has anchored on the cell membrane of E. coli, we performed immunofluorescence assay. HA tag was added to the N-terminal of OprF-CBP so that the recombinant protein OprF-CBP-HA can be specifically recognized by anti-HA antibody. When FITC labeled anti-IgG antibody was used as the secondary antibody and interacted with the primary antibody, green fluorescence could be observed in the cell membrane of E. coli under the fluorescent microscope.




Part 2: The Construction of the Instructor System


All the parts except PpcoA we used to construct E. instructor are from kits in Distribution 2013.They are listed in table2-1.

Table 2-1 Parts from Kits in Distribution 2013

Name Parts Well Short Description
CII BBa_P0153 9A(plate3,2013) Protein
CI BBa_C0051 3A(plate3,2013) Protein
RFP BBa_E1010 18F(plate5,2013) engineered mutant of red fluorescent protein
GFP BBa_E0044 14G(plate5,2013) mGFP mut3b+AAV
PR BBa_R0051 6K(plate5,2013) CI regulated promoter
PRE BBa_R0053 6M(plate5,2013) CII regulated promoter

Assemble all the elements via standard restrict sites:

1. PCR: PpcoA (add EcoRI, XbaI, SpeI and PstI restriction sites)




2. Double digestion: PpcoA PCR products and rbs+CII(LVA)+ter+ter/pSB1C3(BBa_P0153):




3. Colony PCR Validation (primer PpcoA F and PpcoA R):

The plasmid pPcoA-rbs+CII+LVA/pSB1C3 was extracted and sequenced.



4. Double digestion: PpcoA-rbs+CII+LVA/pSB1C3 and rbs+CI+tag-dT:




5. Colony PCR Validation(primer PpcoA F and cI R):

The plasmid PpcoA-rbs+CII+LVA-rbs+CI+tag-dT/pSB1C3 was extracted and sequenced.



6. Double digestion: PCR product of rbs+mRFP+tag and dT/pSB1C3:




7. Colony PCR Validation (primer mRFP F1 and mRFP R1):

The plasmid rbs+mRFP+tag-dT/pSB1C3 were extracted and sequenced.




8. Double digestion: PRE/pMD-19T and rbs+mRFP+tag-dT/pSB1C3:


9. Colony PCR Validation (primer PRE F and mRFP R1):

The plasmid PRE-rbs+mRFP+tag-dT/pMD19-T was extracted and sequenced.



step 2: Transformation of bacteria and colony PCR(primer VF2 and VR)

1. Promoter lambda (cI regulated) with GFP (+LVA) reporter in pSB1A3:

The plasmid PR-rbs+GFP+LVA/pSB1C3 was extracted and sequenced.


2. Double digestion:(3A assembly)

PRE- rbs+mRFP+tag-dT/pMD19-T




3. PpcoA-rbs+CII+LVA-rbs+CI+tag-dT/pSB1C3




4. Colony PCR validation (primer cI F and mRFP R1):

The plasmid PpcoA-rbs+CII+LVA-rbs+CI+tag-dT-PRE-rbs+mRFP+tag-dT/pSB1C3 was extracted and sequenced using primer VF2 and VR.

5. Double digestion PR-rbs+GFP+LVA/pSB1C3




6. PpcoA-rbs+CII+LVA-rbs+CI+tag-dT-PRE-rbs+mRFP+tag-dT/pSB1C3


7. Colony PCR Validation (primer PRE F and GFP R):

The plasmid PpcoA-rbs+CII+LVA-rbs+CI+tag-dT-PRE-rbs+mRFP+tag-dT-PR-rbs+GFP+LVA/pSB1C3 was extracted and sequenced using primer VF2 and VR.




Part 3: The Construction of the Kill Switch

Plasmid construction:

PCR: CI repressor from E. coli phage lambda (+LVA) in pSB1C3(BBa_C0051)was amplified to add RBS using primer:

cI F: 5'–TATGAATTCTCTAGATAAGGAGATATAATGAGCACAAAAAAG-3' and cI R: 5'–TAATCTGCAGACTAGTGCGATCTACACTAGCACTATC-3'


Double digestion:

PCR product of cI+rbs obtained using gel extraction kit and double terminator (dT) in pSB1C3 were applied to double digestion. The reaction systems were listed in table 3-1, 3-2.

Table3-1 Digestion of Fragment (cI+rbs):

Reaction system 50 μl
DNA solution 41 μl
EcoRI 2μl
SpeI 2 μl
10×H 5 μl

Table 3-2 Digestion of pSB1C3-dT:

Reaction system 50 μl
plasmid 41 μl
EcoRI 2μl
SpeI 2 μl
10×M 5 μl

The mixture was incubated in 37℃ for 2h , then gel electrophoresis and gel extraction were performed:

Then, we linked them together at room temperature for 1h. The reaction system listed in table 3-3.

Table 3-3 Reaction System of Ligation

Ligation system 10μl
cI+rbs 4μl
Vector 1μl
T4 Ligase(contain 10x T4 Ligase Buffer) 5μl

Colony PCR validation:

Select monoclonal and applied to PCR:

The positive colone was cultured in LB overnight for plasmid extraction and sequencing.


Assemble all elements one by one using the same method:

Double digestion:

PL lacI (BBa_R0011) in pSB1A3 and pSB1C3-cI+rbs-dT


Colony PCR validation(primer VF2 and cI R):


The plasmid PL lacI-rbs+cI-dT/pSB1C3 was extracted and sequenced.

Double digestion:

PL/pMD-19T and rbs+mRFP+tag PCR products:

Colony PCR validation(primer VF2 and mRFP R1):


Colony PCR validation(primer VF2 and mRFP R1):

The plasmid PL -rbs+mRFP+tag/pSB1C3 was extracted and sequenced.

Double digestion:

PL -rbs+mRFP+tag/pMD19-T and PL lacI-rbs+cI-dT/pSB1C3:


Colony PCR validation (primer VF2 and cI R):

The plasmid PL lacI-rbs+cI-dT- PL -rbs+mRFP+tag/pMD-19T was extracted and sequenced.




Part 4: Measuring of Growth Curve

Step 1:

200μL overnight-cultured bacterial was added to vials containing newly prepared culture medium (containing 100μg/ml Kanamycin). Set one vial with culture medium as blank. All the samples were cultured in the shaking incubator with rotational speed at 200 rpm and temperature at 37℃.

Step 2:

OD600 values was measured using the nucleic acid analyzer. When the OD600 values of culture reached 0.6, 200μL culture was added to a new vial containing 5mL LB, 100μg/ml Kanamycin, 0.1M IPTG and CN-/F-/Cu2+ with different concentration. Set one vial without bacteria as blank. All these samples were cultured in the shaking incubator with rotational speed at 200 rpm and temperature at 37℃.

Step 3:

OD600 of samples from the vials were measured every 30 min.




Part 5: The Standardization of Parts

Materials (used in this part)

The materials we used in this part are listed in table 5-1 and table 5-2

Table 5-1: Bacterial strains and plasmids

Strains and vectors Relevant genotype and characteristics Originate
E. coli DH5α Strains conserved in the lab
pMD18T Vectors TaKaRa Biotechnology (DaLian)Co.,Ltd.
pSB1C3 Vectors iGEM package

Table 5-2: Primers' Name and Sequence

Primer Sequence(5’→3’)
oprF-F G GAATTC GCGGCCGC T TCTAGAG ATGAAACTGAAGAACACC
oprF-R TTT CTGCAG CGGCCGC T ACTAGT ACCAGCCGCCATGAT
ompC-F G GAATTC GCGGCCGC T TCTAGA G ATGCGTCTTGGCTT
ompC-R TGCA CTGCAG CGGCCGC T ACTAGT ATTAGAACTGGTAAACC
RTS-F G GAATTC GCGGCCGC T TCTAGA G TCAGAACGGTTTGG
RTS-R TGCA CTGCAG CGGCCGC T ACTAGT ACTACCGTGATTCATTTC
flA-F G GAATTC GCGGCCGC T TCTAGA G ATGGCTGCCAACAGCAC
flA-R TGCA CTGCAG CGGCCGC T ACTAGT ATCAGCGGGCCTCGACCC
ompC-m-A CCGCTTCTAGAGATGCGTCTTGGCTT
ompC-m-B GGTGTCACCACCGAACTCTGGCAGT
ompC-m-C ACTGCCAGAGTTCGGTGGTGACACC
ompC-m-D GTTACGCCACTAGTAAAGCCTTCAC
ompC-m-E ATTGCAGGTAAGCCAGGGACGGACG
ompC-m-F GTGAAGGCTTTGCTAGTGGCGTAAC
ompC-m-G CGTCCGTCCCTGGCTTACCTGCAAT
ompC-m-H CCGCTACTAGTATTAGAACTGGTAAACC

Step 1: Gene Standardized

P4R amplification with the primers set as the template (pET28a-OprF-CBP/OprF-GS-CBP/cynRTS composite/FLA). The conditions of the reaction were listed in table 5-3.

Table 5-3: The Reaction System to harvest target genes

Components(50μl) Volume(μl)
Template 1.25
dNTPs 2.5
10×LA PCR Buffer 5
LA Tag 0.5
Primer-F(10μmol/L) 2.5
Primer-R(10μmol/L) 2.5
ddH2O 35.75

Step 2: Obliteration of the Illegal Restriction Sites in the Gene

It is necessary to add standard restriction enzyme sites-EcoRI/XbaI/SpeI/PstI to both terminals of each gene. However, the sequence of ompC contains EcoRI, SpeI and PstI restriction site. So we obliterated the restriction sites by site-directed mutagenesis based on overlap extension PCR. Briefly, target mutation (GAATTC→GAGTTC, ACTAGT→GCTAGT , CTGCAG→CTCCAA, ) was introduced into primers (ompC-m-A/B/C/D/E/F/G/H in table 5-2), and the four previous PCR products(ompC-AB, ompC-CD, ompC-EF, ompC-GH) were used as template for the second PCR, and the two previous PCR products(ompC-AD, ompC-EH) were used as template for the third PCR. The final PCR segment with target mutation sites was then cloned into pMD18-T vector for sequencing. The three PCR systems were listed in table 5-4, 5-5, 5-6.

Table 5-4: Reaction System of the 1st PCR

Components(50μl) Volume(μl)
2×Ex Taq Mix 25
ompC-m-A/C/E/G(10μM) 1.5
ompC-m-B/D/F/H(10μM) 1.5
Template 1.5
H2O 20.5

Table 5-5: Reaction System of the 2nd PCR

Components(50μl) Volume(μl)
2×Ex Taq Mix 25
ompC-A/E(10μM) 2.5
ompC-D/H(10μM) 2.5
ompC-AB/EF 5
ompC-CD/GH 5
H2O 10

Table 5-6: Reaction System of 3rd PCR

Components(50μl) Volume(μl)
2×Ex Taq Mix 25
ompC-F(10μM) 2.5
ompC-R(10μM) 2.5
ompC-AD 5
ompC-EH 5
H2O 10

Step 3: Insertion of the Standard Parts into plasmid backbone-pSB1C3

pSB1C3 backbone and the standardized parts was digested with EcoRI and PstI and then retrieved and purified with kits from Omega. pSB1C3 and the standardized parts were then linked together to from new Biobricks.


Step 4 : Submission of New Standardized Parts

After completing all the validation, the four parts were submitted to iGEM official organization in the early October and arrived in New York in 8th Oct. The information was listed in the table 5-7.

Standard biobricks description
pSB1C3-oprF-1(EcoRI PstI) A major outer membrane protein of Pseudomonas aeruginosa functions as a nonspecific porin to allow the passage of small hydrophilic molecules. We cut the protein at 188aa and add a CBP tag to C-terminal with a GS linker between them.
pSB1C3-oprF-2(EcoRI PstI) A major outer membrane protein of Pseudomonas aeruginosa functions as a nonspecific porin to allow the passage of small hydrophilic molecules. We cut the protein at 196aa and add a CBP tag to C-terminal.
pSB1C3-ompC(EcoRI PstI) Outer membrane porin protein C from Escherichia coli BL21(DE3)
pSB1C3-RTS(EcoRI PstI) Cyn operon in Escherichia coli BL21(DE3) (cynR+cynT+cynS) is about cyanate detoxification.
pSB1C3-FLA(EcoRI PstI) Fluorinase enzyme from Streptomyces cattleya catalyzing the formation of a C–F bond by combining S-adenosyl-L-methionine (SAM) and F- to generate 5’-fluoro-5’-deoxyadenosine (5’-FDA) and L-methionine.

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HUST, China