Team:HokkaidoU Japan/Shuffling Kit/How To Use

From 2013.igem.org

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       <h1 id="common-header-title">Maestro E.coli</h1>
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       <h1 id="common-header-title">Maestro <span class="italic"><span class="italic">E. coli</span></span></h1>
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       <h2 id="common-header-subtitle">Optimization Kit</h2>
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       <h2 id="common-header-subtitle">Shuffling Kit</h2>
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       <img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png">
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<h2>What users should prepare</h2>
<h2>What users should prepare</h2>
<p>
<p>
-
   To use the kit, the protein sequence you chose must have specific prefix/suffix which contains a BsaI site and produce overhang. Therefore, users must design a primer to add it. To reduce time and trouble, we automated the design by creating program <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer">"POK-ROK Primer Designer"!</a>
+
   To use the kit, the protein sequence you chose must have specific prefix/suffix which contains a BsaI site and produce overhang. Therefore, users must design a primer to add it. To reduce time and trouble, we automated the design by creating program <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer">"Primer Designer for Maestro"!</a>
</p>
</p>
-
<h2>Promoter Optimization Kit</h2>
+
<h2>Promoter Selector</h2>
<h3>What our kit contains</h3>
<h3>What our kit contains</h3>
<p>
<p>
-
   Our promoter optimization kit consists of 5 different plasmids (table.1). Each has a promoter with a different strength. Downstream of the RBS there is a BsaI site to insert the protein sequence. There is a color expression construct downstream of protein insertion site.(fig 1) Each color is paired with different strength promoter.  The pairings are shown on the table below.
+
   Our Promoter Selector consists of 5 different plasmids (table.1). Each has a promoter with a different strength. Downstream of the RBS there is a BsaI site to insert the protein sequence. There is a color expression construct downstream of protein insertion site (fig.1). Each color is paired with different strength promoter.  The pairings are shown on the table below.
</p>
</p>
<div class="fig fig800">
<div class="fig fig800">
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</tr>
</tr>
<tr>
<tr>
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<td><a href="parts.igem.org/Part:BBa_K1084501">BBa_K1084501</a></td><td><a href="parts.igem.org/Part:BBa_K1084001">BBa_K1084001</a></td><td>Strongest</td><td>amilGFP</td><td>yellowish green</td>
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<td><a href="http://parts.igem.org/Part:BBa_K1084501">BBa_K1084501</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084001">BBa_K1084001</a></td><td>Strongest</td><td>amilGFP</td><td>yellowish green</td>
</tr>
</tr>
<tr>
<tr>
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<td><a href="parts.igem.org/Part:BBa_K1084502">BBa_K1084502</a></td><td><a href="parts.igem.org/Part:BBa_K1084002">BBa_K1084002</a></td><td>Stronger</td><td>aeBlue</td><td>strong blue</td>
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<td><a href="http://parts.igem.org/Part:BBa_K1084502">BBa_K1084502</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084002">BBa_K1084002</a></td><td>Stronger</td><td>aeBlue</td><td>strong blue</td>
</tr>
</tr>
<tr>
<tr>
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<td><a href="parts.igem.org/Part:BBa_K1084503">BBa_K1084503</a></td><td><a href="parts.igem.org/Part:Bba_K1084005">Bba_K1084005</a></td><td>Medium</td><td>amilCP</td><td>Purple</td>
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<td><a href="http://parts.igem.org/Part:BBa_K1084503">BBa_K1084503</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084005">BBa_K1084005</a></td><td>Medium</td><td>amilCP</td><td>Purple</td>
</tr>
</tr>
<tr>
<tr>
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<td><a href="parts.igem.org/Part:BBa_K1084504">BBa_K1084504</a></td><td><a href="parts.igem.org/Part:BBa_K1084009">BBa_K1084009</a></td><td>Weaker</td><td>mRFP</td><td>Pink</td>
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<td><a href="http://parts.igem.org/Part:BBa_K1084504">BBa_K1084504</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084009">BBa_K1084009</a></td><td>Weaker</td><td>mRFP</td><td>Pink</td>
</tr>
</tr>
<tr>
<tr>
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<td><a href="parts.igem.org/Part:BBa_K1084505">BBa_K1084505</a></td><td><a href="parts.igem.org/Part:BBa_K1084010">BBa_K1084010</a></td><td>Weakest</td><td>eforRED</td><td>red</td>
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<td><a href="http://parts.igem.org/Part:BBa_K1084505">BBa_K1084505</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084010">BBa_K1084010</a></td><td>Weakest</td><td>eforRED</td><td>red</td>
</tr>
</tr>
</table>
</table>
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   <div>table.1</div>
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   <div><span class="bold">table.1 Matching list of promoters and their colors.</span></div>
</div>
</div>
<div class="fig fig800">
<div class="fig fig800">
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   <img src="https://static.igem.org/mediawiki/2013/9/95/HokkaidoU_2013_Fig5_new_800.png">
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   <img src="https://static.igem.org/mediawiki/2013/6/60/Fig1_131027_HokkaidoU_2013.png">
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   <div>fig.1</div>
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   <div><span class="bold">fig.1 The matching color for respective promoters.</span></div>
</div>
</div>
<p>
<p>
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   The color expression is induced by IPTG. If you don't need the color expressing construct, you can remove it by using PstI.
+
   The color always expresses. LacZ&alpha; reporter is placed between two BsaI sites (fig.2). The LacZ&alpha; expressing construct will be replaced by chosen sequence using BsaI.
-
  LacZ&alpha; reporter is placed between two BsaI sites(fig.2). The LacZ&alpha; expressing construct will be replaced by chosen sequence using BsaI.
+
</p>
</p>
<div class="fig fig800">
<div class="fig fig800">
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   <img src="https://static.igem.org/mediawiki/2013/9/91/2013_hokkaidou_opti_Fig6_new.png">
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   <img src="https://static.igem.org/mediawiki/2013/c/cc/Fig.2_HokkaidoU_2013.png">
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   <div>fig.2</div>
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   <div><span class="bold">fig.2 Insertion of LacZα reporter between two BsaI sites.</span></div>
</div>
</div>
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<div class="fig fig300">
<div class="fig fig300">
   <img src="https://static.igem.org/mediawiki/2013/2/27/HokkaidoU2013_optimization_Fig7_ver2_400.png">
   <img src="https://static.igem.org/mediawiki/2013/2/27/HokkaidoU2013_optimization_Fig7_ver2_400.png">
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   <div>fig.3</div>
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   <div><span class="bold">fig.3 Digestion by BsaI.</span></div>
</div>
</div>
<p>
<p>
-
   1. Have BsaI site and specific overhang added to your protein sequence (fig.3). PCR with primers designed with <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer">our program</a> should do the trick.
+
   1. Have BsaI site and specific overhang added to your protein sequence (fig.3). PCR with primers designed with <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer">Primer Designer for Maestro</a> should do the trick.
</p>
</p>
<p>
<p>
   2.
   2.
-
   Digest and ligate your protein coding sequence and all our POK kit together(fig.4). This is accomplished by "Golden Gate Assembly" reaction. You can see the recipes of what enzymes and buffer we used in the <a href="2013.igem.org/Team:HokkaidoU_Japan/Notebook">notebook</a>.
+
   Digest and ligate your protein coding sequence and all our Promoter Selector together (fig.4). This is accomplished by "Golden Gate Assembly" reaction. The detailed recipe is shown in Engler (2009)<sup><a href="#cite-1">[1]</a></sup>. All the protein coding sequence will be inserted in the plasmid.
-
  All the protein coding sequence will be inserted in the plasmid.
+
<!-- add more detail -->
</p>
</p>
<div class="fig fig800">
<div class="fig fig800">
-
   <img src="https://static.igem.org/mediawiki/2013/5/51/HokkaidoU_2013Fig8_new_800.png">
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   <img src="https://static.igem.org/mediawiki/2013/a/ac/Fig4_131027_2_HokkaidoU_2013.png">
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   <div>fig.4</div>
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   <div><span class="bold">fig.4 How to insert CDS into protein coding sequence.</span></div>
</div>
</div>
<p>
<p>
-
   3. You should get the construct shown below.(fig.5)
+
   3. You should get the construct shown below after Golden Gate Assembly (fig.5).
</p>
</p>
<div class="fig fig800">
<div class="fig fig800">
-
   <img src="https://static.igem.org/mediawiki/2013/6/66/HokkaidoU_2013_Fig9_new_800.png">
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   <img src="https://static.igem.org/mediawiki/2013/a/a1/Fig5_131027_HokkaidoU_2013.png">
-
   <div>fig.5</div>
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   <div><span class="bold">fig.5 Constructs you can get.</span></div>
</div>
</div>
<p>
<p>
-
   4. Transform the ligated DNA to E. coli, and spread it on plate containing IPTG. Then, you will get colonies with five colors. (fig.6) The colors are paired with the promoters, so you will know which promoter is used instantly!
+
   4. Transform the ligated DNA to <span class="italic">E. coli</span>, and spread it on plate. Then, you will get colonies with five colors easily because you can see 5 colors by naked eyes (fig.6). The colors are paired with the promoters, so you will know what promoter you are using without sequencing all the colonies. You can pick up colonies and have an assay.</p>
-
</p>
+
<div class="fig fig800">
<div class="fig fig800">
-
   <img src="https://static.igem.org/mediawiki/2013/8/88/HokkaidoU2013_optimization_Fig10_new_800.png">
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   <img src="https://static.igem.org/mediawiki/2013/0/07/Assay_new_HokkaidoU_2013.png">
-
   <div>fig.6</div>
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   <div><span class="bold">fig.6 Method after transformation.</span></div>
</div>
</div>
<p>
<p>
-
   5. Pick up the colonies and add to culture. Assay to check the production of protein. When you dont want the colors to be expressed, you can remove the color expressing construct by chosen sequence using Bsa I.(fig.7)
+
    
-
</p>
+
-
<div class="fig fig800">
+
<h2>RBS Selector</h2>
-
  <img src="https://static.igem.org/mediawiki/2013/f/fb/HokkaidoU_2013_Fig11_new_800.png">
+
-
  <div>fig.7</div>
+
-
</div>
+
-
 
+
-
 
+
-
<h2>RBS Optimization Kit</h2>
+
<h3>What our kit contains</h3>
<h3>What our kit contains</h3>
<p>
<p>
-
   Our kit contains tandem RBS (fig.8) and acceptor plasmid (fig.9)
+
   Our kit contains tandem RBS (fig.7) and acceptor plasmid (fig.8).
</p>
</p>
<div class="fig fig800">
<div class="fig fig800">
-
   <img src="https://static.igem.org/mediawiki/2013/2/23/HokkaidoU2013_optimization_Fig12_800.png">
+
   <img src="https://static.igem.org/mediawiki/2013/2/22/RBS_strength_2_HokkaidoU_2013.png">
-
   <div>fig.8</div>
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   <div><span class="bold">fig.7 The sequence of tandem RBS.</span></div>
</div>
</div>
<p>
<p>
-
   In this part, 4 strength levels of RBSs[BBa_K1084101, BBa_ K1084102, BBa_ K1084103, BBa_ K1084104] are connected in tandem. To optimize up to 3 coding sequence expressions in the operon this part has three sets of RBSs with different overhangs are connected together.
+
   In this part, 4 strength levels of RBSs[BBa_K1084101, BBa_ K1084102, BBa_ K1084103, BBa_ K1084104] are connected in tandem. To optimize up to 3 coding sequence expressions in the operon this part has three sets of RBSs with different overhangs and they are connected together.
</p>
</p>
<div class="fig fig800">
<div class="fig fig800">
   <img src="https://static.igem.org/mediawiki/2013/d/d5/HokkaidoU2013_optimization_Fig13_800.png">
   <img src="https://static.igem.org/mediawiki/2013/d/d5/HokkaidoU2013_optimization_Fig13_800.png">
-
   <div>fig.9 This is the acceptor part of the RBS and protein coding region. It has a BsaI site for the parts to be assembled.</div>
+
   <div><span class="bold">fig.8 The acceptor part of the RBS and protein coding region.</span> It has a BsaI site for the parts to be assembled.</div>
</div>
</div>
</p>
</p>
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<p>
<p>
-
   1. Have BsaI site and specific overhang added to your protein sequence. PCR with primers designed with our program should do the trick (fig.9). Also when you want to optimize more than one protein coding sites, add BsaI sites and overhang to them too. Be careful  not to choose the same overhangs.
+
   1. Have BsaI site and specific overhang added to your protein sequence. Again, <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer">Primer Designer for Maestro</a> should help your primers design (fig.9). Also when you want to apply more than one protein coding sites, add BsaI sites and overhang to them too. Be careful  not to choose the same overhangs.
</p>
</p>
<div class="fig fig800">
<div class="fig fig800">
   <img src="https://static.igem.org/mediawiki/2013/e/ef/HokkaidoU2013_optimization_Fig14_800.png">
   <img src="https://static.igem.org/mediawiki/2013/e/ef/HokkaidoU2013_optimization_Fig14_800.png">
-
   <div>fig.10</div>
+
   <div><span class="bold">fig.9 Insertion CDS anf digestion by BsaI.</span></div>
</div>
</div>
<p>
<p>
   2.
   2.
-
   Digest and ligate your protein coding sequence and all ROK kit together. This is accomplished by "Golden Gate Assembly" reaction. You can see the recipes of enzymes and buffer we used in the notebook. DNA fragments will be assembled in the desired order (fig.10).
+
   Digest and ligate your protein coding sequences and all RBS Selector together. This reaction is also accomplished by Golden Gate Assembly. DNA fragments will be assembled in the desired order (fig.10).
</p>
</p>
<div class="fig fig800">
<div class="fig fig800">
   <img src="https://static.igem.org/mediawiki/2013/7/7d/HokkaidoU2013_optimization_Fig15_ver.2_800.png">
   <img src="https://static.igem.org/mediawiki/2013/7/7d/HokkaidoU2013_optimization_Fig15_ver.2_800.png">
-
   <div>fig.10</div>
+
   <div><span class="bold">fig.10 How to use  Golden Gate Assembly.</span></div>
</div>
</div>
<p>
<p>
-
   3. Transform the ligated DNA to E. coli. If you are optimizing three different proteins, you will get 64 different kinds of constructs
+
   3. Transform the ligated DNA to <span class="italic">E. coli</span>. If you are using three different proteins, you will get 64 different kinds of constructs.
</p>
</p>
 +
 +
<p>
 +
We will submit these standard methods as RFC to BioBrick Foundation.
 +
</p>
 +
 +
 +
    <ol class="citation-list">
 +
      <li id="cite-1">C. Engler <span class="italic">et al.</span> Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes (2009) PLoS ONE</li>
 +
    </ol>
 +
 +
<div id="prev-page">
 +
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit"><div class="arrow-div"></div><span>Shuffling Kit Top</span></a>
 +
</div>
 +
 +
<div id="next-page">
 +
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/Primer_Designer"><div class="arrow-div"></div><span>Primer Designer</span></a>
 +
</div>

Latest revision as of 02:49, 29 October 2013

Maestro E. coli

Shuffling Kit

How to use

What users should prepare

To use the kit, the protein sequence you chose must have specific prefix/suffix which contains a BsaI site and produce overhang. Therefore, users must design a primer to add it. To reduce time and trouble, we automated the design by creating program "Primer Designer for Maestro"!

Promoter Selector

What our kit contains

Our Promoter Selector consists of 5 different plasmids (table.1). Each has a promoter with a different strength. Downstream of the RBS there is a BsaI site to insert the protein sequence. There is a color expression construct downstream of protein insertion site (fig.1). Each color is paired with different strength promoter. The pairings are shown on the table below.

Part numberPromoterPromoter strengthPaired proteinProtein color
BBa_K1084501BBa_K1084001StrongestamilGFPyellowish green
BBa_K1084502BBa_K1084002StrongeraeBluestrong blue
BBa_K1084503BBa_K1084005MediumamilCPPurple
BBa_K1084504BBa_K1084009WeakermRFPPink
BBa_K1084505BBa_K1084010WeakesteforREDred
table.1 Matching list of promoters and their colors.
fig.1 The matching color for respective promoters.

The color always expresses. LacZα reporter is placed between two BsaI sites (fig.2). The LacZα expressing construct will be replaced by chosen sequence using BsaI.

fig.2 Insertion of LacZα reporter between two BsaI sites.

How it works

fig.3 Digestion by BsaI.

1. Have BsaI site and specific overhang added to your protein sequence (fig.3). PCR with primers designed with Primer Designer for Maestro should do the trick.

2. Digest and ligate your protein coding sequence and all our Promoter Selector together (fig.4). This is accomplished by "Golden Gate Assembly" reaction. The detailed recipe is shown in Engler (2009)[1]. All the protein coding sequence will be inserted in the plasmid.

fig.4 How to insert CDS into protein coding sequence.

3. You should get the construct shown below after Golden Gate Assembly (fig.5).

fig.5 Constructs you can get.

4. Transform the ligated DNA to E. coli, and spread it on plate. Then, you will get colonies with five colors easily because you can see 5 colors by naked eyes (fig.6). The colors are paired with the promoters, so you will know what promoter you are using without sequencing all the colonies. You can pick up colonies and have an assay.

fig.6 Method after transformation.

RBS Selector

What our kit contains

Our kit contains tandem RBS (fig.7) and acceptor plasmid (fig.8).

fig.7 The sequence of tandem RBS.

In this part, 4 strength levels of RBSs[BBa_K1084101, BBa_ K1084102, BBa_ K1084103, BBa_ K1084104] are connected in tandem. To optimize up to 3 coding sequence expressions in the operon this part has three sets of RBSs with different overhangs and they are connected together.

fig.8 The acceptor part of the RBS and protein coding region. It has a BsaI site for the parts to be assembled.

How to use

1. Have BsaI site and specific overhang added to your protein sequence. Again, Primer Designer for Maestro should help your primers design (fig.9). Also when you want to apply more than one protein coding sites, add BsaI sites and overhang to them too. Be careful not to choose the same overhangs.

fig.9 Insertion CDS anf digestion by BsaI.

2. Digest and ligate your protein coding sequences and all RBS Selector together. This reaction is also accomplished by Golden Gate Assembly. DNA fragments will be assembled in the desired order (fig.10).

fig.10 How to use Golden Gate Assembly.

3. Transform the ligated DNA to E. coli. If you are using three different proteins, you will get 64 different kinds of constructs.

We will submit these standard methods as RFC to BioBrick Foundation.

  1. C. Engler et al. Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes (2009) PLoS ONE
Shuffling Kit Top
Primer Designer

Retrieved from "http://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/How_To_Use"