Difference between revisions of "Team:Jilin China/Construction"

 
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   <p>CONSTRUCTION</p>
 
   <p>CONSTRUCTION</p>
 
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     <td><a href="#paragraph_1" class="clickwave">Protolols</a></td>
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     <td><a href="#paragraph_1" class="clickwave">Golden Gate</a></td>
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     <td><a href="#paragraph_2" class="clickwave">Experiments</a></td>
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     <td><a href="#paragraph_2" class="clickwave">Design</a></td>
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     <td><a href="#paragraph_3" class="clickwave">Development</a></td>
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     <td><a href="#paragraph_3" class="clickwave">Construction</a></td>
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    <td><a href="#paragraph_4" class="clickwave">Procedure</a></td>
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  <li><a href="#paragraph_2">Design</a></li>
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     <div>
 
     <div>
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       <h2>Type IIs Restriction Endonuclease</h2>
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       <h2>Golden Gate Assembly</h2>
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<p>Restriction enzymes can be divided into four categories according to their different enzyme digestion methods, named Type I, Type II, Type III and Type IV. Our commonly used restriction endonucleases, such as EcoRI, belong to type II restriction endonucleases, which recognize specific 4 to 8 nucleotide sequences . The recognized sequence is inverted repeats and the cleavage site is located in the recognition site. After enzymatic cleavage and the cleavage end or blunt end is produced. The cutting diagram is as follows:</p>
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<p>This year, we had hundreds of measurement devices to construct, which means that we need a efficient method to reduce the time and cost of construction. To address this issue, we chose the <b>Golden Gate assembly</b> , which has the advantages of short time-consuming, simple operation and high success rate<sup>[1]</sup>. </p>
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<p>This assembly method is based on the <b>Type IIS restriction endonucleases</b> such as BsaI and BbsI, which differ from Type II restriction enzymes in several ways( figure 1). There are several advantages to use Type IIS restriction endonucleases.</p>
 +
<p>1. The recognition sequences of Type IIS restriction endonucleases comprise two distinct sites, one for DNA binding, the other for DNA cleavage. DNA would be cleaved at a defined distance from their non-palindromic asymmetric recognition sites, creating 4-base overhangs. Therefore, if the Type IIS recognition site is placed outside, 5' to the cleaved end, it will be lost, leading to a <b>‘scar-less’ assembly</b>. This characteristic is widely used to perform in Golden Gate assembly.</p>
 +
<p>2. Type IIS cleavage sites have no inherent sequence-specificity, so the sequence of the overhang they generate varies from one recognition site to another. Fragments produced by Type IIS-digestion of DNA molecules generally have different overhangs, therefore, and will not anneal to one another. However, if the sequences of the overhang are predetermined, by designing them into oligos, then it can be made to complement another and to be directional. Then the DNA fragments can be stitched together in the correct order and orientation in a single ligation<sup>[2]</sup>.</p>
 
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<div class="R2s_animation"><h1>R IIs</h1>
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<div class="R2s_animation"><h1>R IIS</h1>
 
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      <p class="figure">Figure 1. The difference between the Type II restriction edonuclease and Type IIs restriction edonuclease.</p>
  
  
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<p>However, compared with the common Type II restriction enzymes the Type IIS restriction endonuclease is a relatively specific enzyme that cleaves DNA downstream of the recognition site, from the three-dimensional structure analysis of the enzyme. This is due to the fact that the recognition and catalytic regions of the Type IIS restriction enzyme are separated by a polypeptide linker.</p>
 
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<p> The advantage of the TypeII S restriction enzyme is that it has no requirement to a cleavage sequence. The cutting position can be any sequence, and multiple DNA fragments can be assembled through complementary ends. This assembly method is also called GoldenGate Assembly.</p>
 
 
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       <h2>References</h2>
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       <h2>Design of the construction immediate and inserts</h2>
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<p>The goldengate assembly relies on the type IIS restriction enzyme. By adding the recognition site of type IIS enzyme on the plasmid, it will be digest by enzyme and will not appear at the end constructs. The fragment gene of interest contains complementary sticky ends can ultimately be assembled by ligation. </p>
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<p>Firstly, in order to get the measurement device with suitable promoters and different RNA-based thermosensors, we designed the construction immediate (figure 2) in plasmid backbone pSB1C3, which are used to be ligated with many different gene fragments of interest. For the ‘scar-less’ assembly, we add the recognition sites of type IIS enzymes on the construction immediate, which will be digested by enzyme and will not appear at the end constructs. The gene fragments of interest contain complementary sticky ends can ultimately be assembled by simple ligation.</p>
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<p>Commonly used IIS type restriction enzymes are as follows:</p>
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<p class="figure">Figure 2. The construction immediate.</p>
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  <tbody>
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<p>Secondly, our gene fragments of interest such as the DNA fragments of promoters and RNA-based thermosensors contain complementary sticky ends, whitch can ultimately be assembled into the construction immediate by simple ligation.</p>                    
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      <td>Enzyme</td>
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      <td>Recognition Sequence</td>
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<h2>The workflow of the construction and selection</h2>
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      <td>BbsI</td>
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<p>There are <b>stage A</b>, <b>stage B</b> and <b>stage C</b> to construct the measurement devices and selected them by the difference in color. </p>    
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      <td>GAAGAC (2/6)</td>
+
<p>In stage A, the construction immediate will express chromoprotein cjBlue(BBa_K592011) in <i>E.coli</i>. We used Type IIS restriction enzyme BbsI to cut the Part I that contains the sequences of original promoter, cjBlue and double terminator (<b>figure 3, (1)</b>). Then we ligated the inserts of promoters with different expression strength on the plasmid and got the Stage B (<b>figure 3, (2)</b>).</p>
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      <td>BsaI</td>
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      <td>GGTCTC (1/5)</td>
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      <td>BsmBI</td>
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      <td>GCGATG (10/14)</td>
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      <td>Esp3I</td>
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      <td>CGTCTC (1/5)</td>
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<p>We chose <b>BbsI</b> and <b>BsaI</b> type IIS restriction enzymes this year.</p>
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<p>Since we need to build more than 400 components, if we use the traditional assembly method, this will bring a huge amount of work. Due to the low efficiency of traditional assembly, it will seriously delay our experimental process. And Gibson assembly can't guarantee the success rate, and it is not suitable for our large amount of work. Therefore, after the discussion, we finally decide to use GoldenGate Assembly. However, the selection of the correct construct is also a problem, so we designed the following Construction Device to select the correct constructs by visible color changes of the colonies.</p>
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<p>The Construction Device includes a promoter J23104, a pigment protein tspurple, a pigment protein cjblue, a BbsI site, and a BsaI site. J23104+cjblue can be replaced with other promoter sequences by using BbsI restriction enzymes, and tspurple can be replaced with RNA thermosensor sequences using BsaI restriction enzymes. When not replaced, the colonies appear blue due to the expression of cjblue. After the first replacement, the colonies of the correct constructs appeared purple. After the second replacement, the colonies of the correct constructs exhibited their own milky white color. Color changes can refer to the following table:</p>
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<p>If the previous steps were successful conducted, the plasmid could express chromoprotein tsPurple(BBa_K1033906) rather than cjBlue in stage B. At this time, we chose the purple monocolony and extracted its plasmids. After the correct sequencing, then we conducted the next step.</p>
 +
    <p>In the next step, we used Type IIS restriction enzyme BsaI to cut the Part II that contains the sequences of RBS, tsPurple and double terminator (<b>figure 3, (3)</b>), and replaced this region with the inserts of different RNA-based thermosensors (<b>figure 3, (4)</b>).</p>
 +
    <p>If the previous steps were successful conducted, the plasmid could express superfold GFP rather than tsPurple. After the correct sequencing, we got different measurement devices. If they did not have expression intensity that is easy to detect, we would replace the promoter until the appropriate intensity was reached.</p>
 +
                <center><img src="https://static.igem.org/mediawiki/2018/6/64/T--Jilin_China--Construction-Construction_procedure.svg" width="64%" /></center>
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<p class="figure">Figure 3. The steps of construction of measurement devices.</p>
 
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       <h2>References</h2>
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       <h2>The procedure of Golden Gate assembly</h2>
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       <ul>
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       <p>We followed three steps to assemble many measurement devices as follows:</p>
−
      <li id="r1">Dai H, Wang Y, Lu X, et al. Chimeric antigen receptors modified T-cells for cancer therapy[J]. JNCI: Journal of the National Cancer Institute, 2016, 108(7).</li>
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      <h3>1.Annealed oligos</h3>
−
      <li>aaaaaa</li>
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      <p>This step could be used to add any short DNA fragment to a plasmid. Most of the thermosensors we designed are 40~80bp, so we designed top oligos with the sequences of thermosensors and with the bottom oligos, which would be the reverse compliments so that they could anneal. We made sure our annealing products contain the cohesive end bases to complement the overhangs on the construction device (Figure 4).</p>
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      <li>aaaaaa</li>
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      <center><img src="https://static.igem.org/mediawiki/2018/0/0a/T--Jilin_China--Construction-anneal.svg" width="70%" /></center>
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                <p class="figure">Figure 4. Diagram of Annealed oligos</p>
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      <h3>2. Phosphorylation</h3>
 +
      <p>After annealing, the 5' phosphorylation of DNA was required for the subsequent ligation. We phosphorylated fragments with T4 Polynucleotide kinase, which could catalyze the transfer and exchange of P from the γ position of ATP to the 5'-hydroxyl terminus of polynucleotides and 3'-monophosphates (Figure 5).</p>
 +
      <center><img src="https://static.igem.org/mediawiki/2018/c/c0/T--Jilin_China--Construction-phosphorylation.svg" width="70%" /></center>
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                <p class="figure">Figure 5. Diagram of Phosphorylation</p>
 +
       <h3>3.Digestion and Ligation</h3>
 +
      <p>We chose Type IIS restriction enzyme to digest the plasmid and used T4 DNA ligase to assemble DNA fragments we had phosphorylated before (figure 6).</p>
 +
      <center><img src="https://static.igem.org/mediawiki/2018/e/e7/T--Jilin_China--Construction-GG.svg" width="70%" /></center>
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                <p class="figure">Figure 6. Diagram of Digestion and Ligation</p>    </div>
 
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      <h2>Type IIs Restriction Endonuclease</h2>
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<p>Restriction enzymes can be divided into four categories according to their different enzyme digestion methods, named Type I, Type II, Type III and Type IV. Our commonly used restriction endonucleases, such as EcoRI, belong to type II restriction endonucleases, which recognize specific 4 to 8 nucleotide sequences . The recognized sequence is inverted repeats and the cleavage site is located in the recognition site. After enzymatic cleavage and the cleavage end or blunt end is produced. The cutting diagram is as follows:</p>
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</div>
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<div class="R2s_animation"><h1>R IIs</h1>
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opacity: 1;
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opacity: 0;
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    <polyline class="i cut" points="365.12 65.73 365.12 101.51 477.12 101.51 477.12 134.23"/>
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<!--预警结束[Warning End]-->
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<p>However, compared with the common Type II restriction enzymes the Type IIS restriction endonuclease is a relatively specific enzyme that cleaves DNA downstream of the recognition site, from the three-dimensional structure analysis of the enzyme. This is due to the fact that the recognition and catalytic regions of the Type IIS restriction enzyme are separated by a polypeptide linker.</p>
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<p> The advantage of the TypeII S restriction enzyme is that it has no requirement to a cleavage sequence. The cutting position can be any sequence, and multiple DNA fragments can be assembled through complementary ends. This assembly method is also called GoldenGate Assembly.</p>
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      <h2>References</h2>
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<p>The goldengate assembly relies on the type IIS restriction enzyme. By adding the recognition site of type IIS enzyme on the plasmid, it will be digest by enzyme and will not appear at the end constructs. The fragment gene of interest contains complementary sticky ends can ultimately be assembled by ligation. </p>
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<p>Commonly used IIS type restriction enzymes are as follows:</p>
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<table width="200" border="1">
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      <td>Enzyme</td>
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      <td>Recognition Sequence</td>
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      <td>BbsI</td>
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      <td>GAAGAC (2/6)</td>
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      <td>BsaI</td>
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      <td>GGTCTC (1/5)</td>
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      <td>Esp3I</td>
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      <td>SapI</td>
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<p>We chose <b>BbsI</b> and <b>BsaI</b> type IIS restriction enzymes this year.</p>
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<p>Since we need to build more than 400 components, if we use the traditional assembly method, this will bring a huge amount of work. Due to the low efficiency of traditional assembly, it will seriously delay our experimental process. And Gibson assembly can't guarantee the success rate, and it is not suitable for our large amount of work. Therefore, after the discussion, we finally decide to use GoldenGate Assembly. However, the selection of the correct construct is also a problem, so we designed the following Construction Device to select the correct constructs by visible color changes of the colonies.</p>
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<p>The Construction Device includes a promoter J23104, a pigment protein tspurple, a pigment protein cjblue, a BbsI site, and a BsaI site. J23104+cjblue can be replaced with other promoter sequences by using BbsI restriction enzymes, and tspurple can be replaced with RNA thermosensor sequences using BsaI restriction enzymes. When not replaced, the colonies appear blue due to the expression of cjblue. After the first replacement, the colonies of the correct constructs appeared purple. After the second replacement, the colonies of the correct constructs exhibited their own milky white color. Color changes can refer to the following table:</p>
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<img src="https://static.igem.org/mediawiki/2018/2/25/T--Jilin_China--Parts--Composite_Part--color_table.png" align="center"/>
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       <h2>References</h2>
 
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       <li id="r1">Dai H, Wang Y, Lu X, et al. Chimeric antigen receptors modified T-cells for cancer therapy[J]. JNCI: Journal of the National Cancer Institute, 2016, 108(7).</li>
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       <li id="r1">[1]Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability[J]. PloS one, 2008, 3(11): e3647.</li>
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       <li>aaaaaa</li>
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       <li>[2]Weber E, Gruetzner R, Werner S, et al. Assembly of designer TAL effectors by Golden Gate cloning[J]. PloS one, 2011, 6(5): e19722.</li>
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Latest revision as of 03:05, 18 October 2018

CONSTRUCTION


Golden Gate Design Construction Procedure

Construction

  • Golden Gate Assembly

    This year, we had hundreds of measurement devices to construct, which means that we need a efficient method to reduce the time and cost of construction. To address this issue, we chose the Golden Gate assembly , which has the advantages of short time-consuming, simple operation and high success rate[1].

    This assembly method is based on the Type IIS restriction endonucleases such as BsaI and BbsI, which differ from Type II restriction enzymes in several ways( figure 1). There are several advantages to use Type IIS restriction endonucleases.

    1. The recognition sequences of Type IIS restriction endonucleases comprise two distinct sites, one for DNA binding, the other for DNA cleavage. DNA would be cleaved at a defined distance from their non-palindromic asymmetric recognition sites, creating 4-base overhangs. Therefore, if the Type IIS recognition site is placed outside, 5' to the cleaved end, it will be lost, leading to a ‘scar-less’ assembly. This characteristic is widely used to perform in Golden Gate assembly.

    2. Type IIS cleavage sites have no inherent sequence-specificity, so the sequence of the overhang they generate varies from one recognition site to another. Fragments produced by Type IIS-digestion of DNA molecules generally have different overhangs, therefore, and will not anneal to one another. However, if the sequences of the overhang are predetermined, by designing them into oligos, then it can be made to complement another and to be directional. Then the DNA fragments can be stitched together in the correct order and orientation in a single ligation[2].

    R II

    R IIS

    R IIs

    Figure 1. The difference between the Type II restriction edonuclease and Type IIs restriction edonuclease.

  • Design of the construction immediate and inserts

    Firstly, in order to get the measurement device with suitable promoters and different RNA-based thermosensors, we designed the construction immediate (figure 2) in plasmid backbone pSB1C3, which are used to be ligated with many different gene fragments of interest. For the ‘scar-less’ assembly, we add the recognition sites of type IIS enzymes on the construction immediate, which will be digested by enzyme and will not appear at the end constructs. The gene fragments of interest contain complementary sticky ends can ultimately be assembled by simple ligation.

    Figure 2. The construction immediate.

    Secondly, our gene fragments of interest such as the DNA fragments of promoters and RNA-based thermosensors contain complementary sticky ends, whitch can ultimately be assembled into the construction immediate by simple ligation.

  • The workflow of the construction and selection

    There are stage A, stage B and stage C to construct the measurement devices and selected them by the difference in color.

    In stage A, the construction immediate will express chromoprotein cjBlue(BBa_K592011) in E.coli. We used Type IIS restriction enzyme BbsI to cut the Part I that contains the sequences of original promoter, cjBlue and double terminator (figure 3, (1)). Then we ligated the inserts of promoters with different expression strength on the plasmid and got the Stage B (figure 3, (2)).

    If the previous steps were successful conducted, the plasmid could express chromoprotein tsPurple(BBa_K1033906) rather than cjBlue in stage B. At this time, we chose the purple monocolony and extracted its plasmids. After the correct sequencing, then we conducted the next step.

    In the next step, we used Type IIS restriction enzyme BsaI to cut the Part II that contains the sequences of RBS, tsPurple and double terminator (figure 3, (3)), and replaced this region with the inserts of different RNA-based thermosensors (figure 3, (4)).

    If the previous steps were successful conducted, the plasmid could express superfold GFP rather than tsPurple. After the correct sequencing, we got different measurement devices. If they did not have expression intensity that is easy to detect, we would replace the promoter until the appropriate intensity was reached.

    Figure 3. The steps of construction of measurement devices.

  • The procedure of Golden Gate assembly

    We followed three steps to assemble many measurement devices as follows:

    1.Annealed oligos

    This step could be used to add any short DNA fragment to a plasmid. Most of the thermosensors we designed are 40~80bp, so we designed top oligos with the sequences of thermosensors and with the bottom oligos, which would be the reverse compliments so that they could anneal. We made sure our annealing products contain the cohesive end bases to complement the overhangs on the construction device (Figure 4).

    Figure 4. Diagram of Annealed oligos

    2. Phosphorylation

    After annealing, the 5' phosphorylation of DNA was required for the subsequent ligation. We phosphorylated fragments with T4 Polynucleotide kinase, which could catalyze the transfer and exchange of P from the γ position of ATP to the 5'-hydroxyl terminus of polynucleotides and 3'-monophosphates (Figure 5).

    Figure 5. Diagram of Phosphorylation

    3.Digestion and Ligation

    We chose Type IIS restriction enzyme to digest the plasmid and used T4 DNA ligase to assemble DNA fragments we had phosphorylated before (figure 6).

    Figure 6. Diagram of Digestion and Ligation

  • References

    • [1]Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability[J]. PloS one, 2008, 3(11): e3647.
    • [2]Weber E, Gruetzner R, Werner S, et al. Assembly of designer TAL effectors by Golden Gate cloning[J]. PloS one, 2011, 6(5): e19722.