Difference between revisions of "Team:HUST-China/Basic Part"

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<h2> Basic Parts</h2>
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<title>Team:HUST-China:Results</title>
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<h4>Note</h4>
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<p>In order to be considered for the <a href="https://2015.igem.org/Judging/Awards#SpecialPrizes">Best New Basic Part award</a>, you must fill out this page. Please give links to the Registry entries for the Basic parts you have made. Please see the Registry's <a href="http://parts.igem.org/Help:Parts#Basic_and_Composite_Parts"> Help:Parts page</a> for more information on part types.</p>
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A <b>basic part</b> is a functional unit of DNA that cannot be subdivided into smaller component parts. <a href="http://parts.igem.org/wiki/index.php/Part:BBa_R0051">BBa_R0051</a> is an example of a basic part, a promoter regulated by lambda cl.
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<p>Most genetically-encoded functions have not yet been converted to BioBrick parts. Thus, there are <b>many</b> opportunities to find new, cool, and important genetically encoded functions, and refine and convert the DNA encoding these functions into BioBrick standard biological parts. </p>
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<h4 align="center" style="color:white"><b>click it~</b></h4>
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        <p> This year, we iGEM_HUST-China team constructed a darkness induction system based on yeast two hybrid, which is used to promote the expression of downstream working proteins: Si-tag and Mcfp-3.<br>
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With the basic or composite parts we used in our Euk.cement project, together with some former parts in pools we improved by mutating correction of some mistakes, this year we totally constructed, sequenced, fully documented and submitted 26 parts.
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<br><br>
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Following documented our parts submitted.
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    </p>
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      <h3 style="color:black" align="left"><b> Parts in Our Euk.Cement Project</b></h3><br>
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            <h4 style="color:black" align="left"><b> 1. Basic Parts : Light Control Parts of Darkness Induction System</b></h4><br>
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 +
        <table border="1">
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            <tr>
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<th>Part Number</th>
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<th>Description</th>
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<th>Nickname</th>
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</tr>
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<tr>
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<td><a href="http://parts.igem.org/Part:BBa_K1592005">BBa_K1592005</a></td>
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<td>GalBD-CRY2 Fusion for Yeast-Two-Hybrid</td>
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<td>BD-CRY2</td>
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</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592006">BBa_K1592006</a></td>
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<td>GalAD-CIB1 Fusion for Yeast-Two-Hybrid</td>
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<td>AD-CIB1</td>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592015">BBa_K1592015</a></td>
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<td>photoreceptor cryptochrome 2</td>
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<td>CRY2</td>
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</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592016">BBa_K1592016</a></td>
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<td>a basic helix-loop-helix protein</td>
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<td>CIB1</td>
 +
</tr>
 +
</table>
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<p> Our light-control system is based on a Yeast-Two-Hybrid system. Cryptochrome 2 (CRY2) is a blue light stimulated photoreceptor, when exposed to blue light, CRY2 fused with BD would interact with CIB1 fused with AD ,this CRY2-CIB1 interaction will bring AD and BD of Gal4 gene together to activate the downstream negative control parts.<br>
 +
We verified our Light-control system by measuring β-galactosidase activity that induced by Gal4.
 +
  </p>
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          <h4 style="color:black" align="left"><b> Composite part</b></h4><br>
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 +
<table border="1">
 +
            <tr>
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<th>Part Number</th>
 +
<th>Description</th>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592018">BBa_K1592018</a></td>
 +
<td> Pgal1+rox1+cyc1_terminator </td>
 +
</tr>
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</table>
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<p> This composite part BBa_K1592018 consists of a Pgal1 promoter, a ROX1 inhibitor gene that is controlled by Pgal1 promoter and a terminator, The Pgal1 promoter can be activated by CRY2-CIB1 light-control parts through UAS. Expressed ROX1 protein will inhibit following working parts from expression.
 +
</p>
 +
<div>
 +
    <p>BBa_K1592018</p>
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<img class="picture" src="https://static.igem.org/mediawiki/2015/8/88/HUST_part3.png">
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</div><br>
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<div>
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    <img class="picture" src="https://static.igem.org/mediawiki/2015/3/33/HUST_result1.jpg">
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            <h4 style="color:black" align="left"><b> 2. Basic Parts: Secrete and Surface Display</b></h4><br>
 +
            <table border="1">
 +
            <tr>
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<th>Part Number</th>
 +
<th>Description</th>
 +
<th>Nickname</th>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592000">BBa_K1592000</a></td>
 +
<td>LIP2 prepro(signal peptide)</td>
 +
<td>LIP2 prepro</td>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592002">BBa_K1592002</a></td>
 +
<td>Yarrowia lipolytica cell wall protein 3</td>
 +
<td>YLcwp3</td>
 +
</tr>
 +
</table>
 +
<p> LIP2 prepro is a signal peptide. When fused to the N-terminal of target protein, the expression products will be secreted out of cell.
 +
<br><br>
 +
YLcwp3, also known as an anchor domain, is a yeast cell wall protein. When fused to the C-terminal of target protein, the expression products will be displayed on the cell wall.
 +
</p>
 +
        <h4 style="color:black" align="left"><b> 3. Basic Parts: Silica binding proteins (Si-tag) Collection</b></h4><br>
 +
          <table border="1">
 +
            <tr>
 +
<th>Part Number</th>
 +
<th>Description</th>
 +
<th>Nickname</th>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592007">BBa_K1592007</a></td>
 +
<td>LIP prepro + E. coli ribosomal protein L2 (1-60) + YLcwp3 Fusion</td>
 +
<td>Si-tag1-his</td>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592008">BBa_K1592008</a></td>
 +
<td>LIP prepro + E. coli ribosomal protein L2 (61-202) + YLcwp3 Fusion</td>
 +
<td>Si-tag2-his</td>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592009">BBa_K1592009</a></td>
 +
<td>LIP prepro + E. coli ribosomal protein L2 (203-273) + YLcwp3 Fusion</td>
 +
<td>Si-tag3-his</td>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592010">BBa_K1592010</a></td>
 +
<td>LIP2 prepro + E. coli ribosomal protein L2 (1-202)+ YLcwp3 Fusion</td>
 +
<td>ST12</td>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592011">BBa_K1592011</a></td>
 +
<td>LIP prepro + E. coli ribosomal protein L2 (61-273) + YLcwp3 Fusion</td>
 +
<td>ST23</td>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592012">BBa_K1592012</a></td>
 +
<td>LIP prepro + E. coli ribosomal protein L2 (1-60,203-273) + YLcwp3 Fusion</td>
 +
<td>ST13</td>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592013">BBa_K1592013</a></td>
 +
<td>LIP prepro + E. coli ribosomal protein L2 (1-60,GS linker,202-273) + YLcwp3</td>
 +
<td>ST1L3-his</td>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592014">BBa_K1592014</a></td>
 +
<td>LIP prepro + E. coli ribosomal protein L2 (1-273) + YLcwp3</td>
 +
<td>ST123</td>
 +
</tr>
 +
</table>
 +
<br><p> This collection consists of several silica binding proteins (Si-tag) that can be surface displayed. Si-tag is 50S ribosomal protein L2 in the genome of E.coli, which was found to bind tightly to silica particles. The full length 50S ribosomal protein L2 protein consists of three domains showing different silica binding binding intensity. We constructed 8 different Si-tag isoforms with different truncations and domain combinations of this 50S ribosomal protein L2. We further added LIP prepro and YLcwp3(see below) to the terminals of Si-tag, thus Si-tag can be secreted and surface displayed on the cell wall.Finally we tested their silica binding intensity in situ.
 +
<p><a href="https://2015.igem.org/Team:HUST-China/Results#4">Click HERE or part number to see more details.</a></p>
 +
<div>
 +
          <img class="picture" src="https://static.igem.org/mediawiki/2015/8/8e/HUST_part2.png">
 +
          </div>
 +
<h4 style="color:black" align="left"><b> 4. Basic Parts: Flocculating Protein Mcfp3</b></h4><br>
 +
<table border="1">
 +
            <tr>
 +
<th>Part Number</th>
 +
<th>Description</th>
 +
<th>Abbreviation</th>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592001">BBa_K1592001</a></td>
 +
<td>Mytilus californianus foot protein 3(Mcfp3) variant 3</td>
 +
<td>Mcfp3</td>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592003">BBa_K1592003</a></td>
 +
<td>Mcfp3 with LIP2 prepro</td>
 +
<td>LIP-Mcfp</td>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592017">BBa_K1592017</a></td>
 +
<td>Mcfp3 with XPR2 pre</td>
 +
<td>XPR2-Mcfp</td>
 +
</tr>
 +
</table>
 +
         
 +
<p> Mcfp-3 is a flocculating protein secreted by Mytilus californianus (mussels). The protein is of significance to the formation of filopodia to help mussels permanently or temporarily tether to the surface of solid surface of reef or ships.
 +
<br>
 +
LIP2 and XPR2 are signal peptides. Signal peptide is added to the top of Mcfp3 sequence, thus Mcfp-3 can be secreted out of cell.
 +
            </p>
 +
<div>
 +
          <img style="width=150px;height=350px":class="picture" src="https://static.igem.org/mediawiki/2015/c/cc/Part4-hust.jpeg">
 +
          </div>
 +
<div>
 +
          <img class="picture" src="https://static.igem.org/mediawiki/2015/a/aa/Figure_legend-hust.jpeg">
 +
          </div>
 +
<h4 style="color:black" align="left"><b>Composite Part </b></h4><br>
 +
<table border="1">
 +
            <tr>
 +
<th>Part Number</th>
 +
<th>Description</th>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592019">BBa_K1592019</a></td>
 +
<td> Panb1+XPR2 pre-Mcfp3 </td>
 +
</tr>
 +
</table>
 +
<br><p> This composite part BBa_K1592019 consists of a Panb1 promoter, and a XPR2 pre signal peptide initiated Mcfp3 gene. With this part, the Mcfp-3 gene can be expressed and The product flocculating protein will finally secreted outside cell.</p><br>
 +
<div>
 +
    <p>BBa_K1592018</p>
 +
<img class="picture" src="https://static.igem.org/mediawiki/2015/9/90/HUST_part4.png">
 +
</div><br>
 +
<h4 style="color:black" align="left"><b>5.Basic Parts: Promoter hp4d </b></h4><br>
 +
<table border="1">
 +
            <tr>
 +
<th>Part Number</th>
 +
<th>Description</th>
 +
<th>Nickname</th>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592004">BBa_K1592004</a></td>
 +
<td>promoter hp4d</td>
 +
<td>Php4d</td>
 +
</tr>
 +
</table>
 +
<br><p> Promoter hp4d is a recombinant promoter which can strongly promote gene expression in any culture medium. The gene promoted by Php4d usually expresses at the early stage of stabilization.
 +
</p><br>
 +
<h3 style="color:black" align="left"><b>Parts Improvement </b></h3><br>
 +
<h4 style="color:black" align="left"><b> Basic Parts: Improved Ptrp Promoter </b></h4><br>
 +
<table border="1">
 +
            <tr>
 +
<th>Part Number</th>
 +
<th>Description</th>
 +
<th>Nickname</th>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592020">BBa_K1592020</a></td>
 +
<td>Ptrp mutant1</td>
 +
<td>Ptrp1</td>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592021">BBa_K1592021</a></td>
 +
<td>Ptrp mutant2</td>
 +
<td>Ptrp2</td>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592022">BBa_K1592022</a></td>
 +
<td>Ptrp mutant3</td>
 +
<td>Ptrp3</td>
 +
</tr>
 +
</table>
 +
<br><p> Ptrp is a promoter of  trp operon exists in parts pools with cat. Number BBa_K191007. It will be repressed by trpR and LovTAP. This year, We improved this BBa_K191007 biobrick. We removed the illegal restriction sites without affecting its function by site-directed mutagenesis to fulfill the requirement of RFC10. Finally we constructed three ptrp mutants, named Ptrp mutant1, Ptrp mutant2, and Ptrp mutant3.
 +
</p><br>
 +
<h4 style="color:black" align="left"><b> Composite Part </b></h4><br>
 +
<table border="1">
 +
            <tr>
 +
<th>Part Number</th>
 +
<th>Description</th>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592023">BBa_K1592023</a></td>
 +
<td>Ptrp mutant1+RBS+GFP</td>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592024">BBa_K1592024</a></td>
 +
<td>Ptrp mutant2+RBS+GFP</td>
 +
</tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1592025">BBa_K1592025</a></td>
 +
<td>Ptrp mutant3+RBS+GFP</td>
 +
</tr>
 +
</table>
 +
<br><p> These composite parts consist of a improved Ptrp promoter, a RBS and a GFP fluorescent report gene. With this parts, the promoting function of Ptrp promoter can be evaluated.</p><br>
 +
<div>
 +
    <p>BBa_K1592018</p>
 +
<img class="picture" src="https://static.igem.org/mediawiki/2015/c/cf/HUST_part5.png">
 +
</div>
 +
<br><h4> These Ptrp promoters were not really applied in our project. However, we successfully found the defects of existed biobrick in pools and improved it to fulfill the basic requirement of biobrick property. This parts improvement work is a required achievement in judging<br><br><br>
 +
</h4>
 +
  
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Latest revision as of 01:16, 18 September 2015

Team:HUST-China:Results


click it~


This year, we iGEM_HUST-China team constructed a darkness induction system based on yeast two hybrid, which is used to promote the expression of downstream working proteins: Si-tag and Mcfp-3.
With the basic or composite parts we used in our Euk.cement project, together with some former parts in pools we improved by mutating correction of some mistakes, this year we totally constructed, sequenced, fully documented and submitted 26 parts.

Following documented our parts submitted.

Parts in Our Euk.Cement Project


1. Basic Parts : Light Control Parts of Darkness Induction System


Part Number Description Nickname
BBa_K1592005 GalBD-CRY2 Fusion for Yeast-Two-Hybrid BD-CRY2
BBa_K1592006 GalAD-CIB1 Fusion for Yeast-Two-Hybrid AD-CIB1
BBa_K1592015 photoreceptor cryptochrome 2 CRY2
BBa_K1592016 a basic helix-loop-helix protein CIB1

Our light-control system is based on a Yeast-Two-Hybrid system. Cryptochrome 2 (CRY2) is a blue light stimulated photoreceptor, when exposed to blue light, CRY2 fused with BD would interact with CIB1 fused with AD ,this CRY2-CIB1 interaction will bring AD and BD of Gal4 gene together to activate the downstream negative control parts.
We verified our Light-control system by measuring β-galactosidase activity that induced by Gal4.

Composite part


Part Number Description
BBa_K1592018 Pgal1+rox1+cyc1_terminator

This composite part BBa_K1592018 consists of a Pgal1 promoter, a ROX1 inhibitor gene that is controlled by Pgal1 promoter and a terminator, The Pgal1 promoter can be activated by CRY2-CIB1 light-control parts through UAS. Expressed ROX1 protein will inhibit following working parts from expression.

BBa_K1592018


2. Basic Parts: Secrete and Surface Display


Part Number Description Nickname
BBa_K1592000 LIP2 prepro(signal peptide) LIP2 prepro
BBa_K1592002 Yarrowia lipolytica cell wall protein 3 YLcwp3

LIP2 prepro is a signal peptide. When fused to the N-terminal of target protein, the expression products will be secreted out of cell.

YLcwp3, also known as an anchor domain, is a yeast cell wall protein. When fused to the C-terminal of target protein, the expression products will be displayed on the cell wall.

3. Basic Parts: Silica binding proteins (Si-tag) Collection


Part Number Description Nickname
BBa_K1592007 LIP prepro + E. coli ribosomal protein L2 (1-60) + YLcwp3 Fusion Si-tag1-his
BBa_K1592008 LIP prepro + E. coli ribosomal protein L2 (61-202) + YLcwp3 Fusion Si-tag2-his
BBa_K1592009 LIP prepro + E. coli ribosomal protein L2 (203-273) + YLcwp3 Fusion Si-tag3-his
BBa_K1592010 LIP2 prepro + E. coli ribosomal protein L2 (1-202)+ YLcwp3 Fusion ST12
BBa_K1592011 LIP prepro + E. coli ribosomal protein L2 (61-273) + YLcwp3 Fusion ST23
BBa_K1592012 LIP prepro + E. coli ribosomal protein L2 (1-60,203-273) + YLcwp3 Fusion ST13
BBa_K1592013 LIP prepro + E. coli ribosomal protein L2 (1-60,GS linker,202-273) + YLcwp3 ST1L3-his
BBa_K1592014 LIP prepro + E. coli ribosomal protein L2 (1-273) + YLcwp3 ST123

This collection consists of several silica binding proteins (Si-tag) that can be surface displayed. Si-tag is 50S ribosomal protein L2 in the genome of E.coli, which was found to bind tightly to silica particles. The full length 50S ribosomal protein L2 protein consists of three domains showing different silica binding binding intensity. We constructed 8 different Si-tag isoforms with different truncations and domain combinations of this 50S ribosomal protein L2. We further added LIP prepro and YLcwp3(see below) to the terminals of Si-tag, thus Si-tag can be secreted and surface displayed on the cell wall.Finally we tested their silica binding intensity in situ.

Click HERE or part number to see more details.

4. Basic Parts: Flocculating Protein Mcfp3


Part Number Description Abbreviation
BBa_K1592001 Mytilus californianus foot protein 3(Mcfp3) variant 3 Mcfp3
BBa_K1592003 Mcfp3 with LIP2 prepro LIP-Mcfp
BBa_K1592017 Mcfp3 with XPR2 pre XPR2-Mcfp

Mcfp-3 is a flocculating protein secreted by Mytilus californianus (mussels). The protein is of significance to the formation of filopodia to help mussels permanently or temporarily tether to the surface of solid surface of reef or ships.
LIP2 and XPR2 are signal peptides. Signal peptide is added to the top of Mcfp3 sequence, thus Mcfp-3 can be secreted out of cell.

Composite Part


Part Number Description
BBa_K1592019 Panb1+XPR2 pre-Mcfp3

This composite part BBa_K1592019 consists of a Panb1 promoter, and a XPR2 pre signal peptide initiated Mcfp3 gene. With this part, the Mcfp-3 gene can be expressed and The product flocculating protein will finally secreted outside cell.


BBa_K1592018


5.Basic Parts: Promoter hp4d


Part Number Description Nickname
BBa_K1592004 promoter hp4d Php4d

Promoter hp4d is a recombinant promoter which can strongly promote gene expression in any culture medium. The gene promoted by Php4d usually expresses at the early stage of stabilization.


Parts Improvement


Basic Parts: Improved Ptrp Promoter


Part Number Description Nickname
BBa_K1592020 Ptrp mutant1 Ptrp1
BBa_K1592021 Ptrp mutant2 Ptrp2
BBa_K1592022 Ptrp mutant3 Ptrp3

Ptrp is a promoter of trp operon exists in parts pools with cat. Number BBa_K191007. It will be repressed by trpR and LovTAP. This year, We improved this BBa_K191007 biobrick. We removed the illegal restriction sites without affecting its function by site-directed mutagenesis to fulfill the requirement of RFC10. Finally we constructed three ptrp mutants, named Ptrp mutant1, Ptrp mutant2, and Ptrp mutant3.


Composite Part


Part Number Description
BBa_K1592023 Ptrp mutant1+RBS+GFP
BBa_K1592024 Ptrp mutant2+RBS+GFP
BBa_K1592025 Ptrp mutant3+RBS+GFP

These composite parts consist of a improved Ptrp promoter, a RBS and a GFP fluorescent report gene. With this parts, the promoting function of Ptrp promoter can be evaluated.


BBa_K1592018


These Ptrp promoters were not really applied in our project. However, we successfully found the defects of existed biobrick in pools and improved it to fulfill the basic requirement of biobrick property. This parts improvement work is a required achievement in judging