Team:Biwako Nagahama/Material & Method

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= <h2>Material & Method</h2> =
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== <html>
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<ul>
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<li><a href="https://2013.igem.org/Team:Biwako_Nagahama/general protocol"><h2>General protocol</h2></a></li>
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<ul>
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= <h2>AgRePaper&E.coli-ink</h2> =
 
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Cellulose is used as raw material for paper, so our team experimented various ways to increase the amount of cellulose produced by agrobacterium and using it to make papers. For this we developed the different parts to insert into the system of agrobacterium. Among them are the genes used for expression of the curdlan. Similarly, genetic parts in order to increase the expression of the cellulose, along with the agrobacterium type binary vector were also developed . We are also working on recycling the produced paper by degrading the cellulose to D-Glucose using various enzymes. We worked for the preparation of the biological ink using the sperm whale's cells by genetically modification to increase amount of myoglobin. Then, we observed the change on the color of the product by altering the formation of myoglobin and the production amount of myoglobin with the insertion of T7 promoter to the cell system.
 
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== <h2>Agrepaper</h2> ==
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</html> ==
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=== <h2>PROBLEM PRESENTATION</h2> ===
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<p>①There are variety of issues that advanced in from today.
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For example Grobal warming,ozone layer destruction,biological extinction,an energy problem,desertification ,a crisis of water resources.There issues are coused by deforestation to make papers.</p>
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<p>②How to solve it.</p>
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<p>③Environment issues can solve if not to use wood as well as burdon the environment.
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We need to know “What is the paper?” to think the method.</p>
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<p>④The main component of paper is cellulose and hemicellulose.</p>
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<p>⑤We were confirmed by Nakasima’s experiments that Agrobacterium make cellulose.
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Agrobacterium can’t make hemicellulose but cardlan Agrobacterium made has potential instead of hemicellulose, Ootaki perform confimatory experiment .As a result cardlan is able to instead of hemicellulose.
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</p>
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=== <h2>中島の実験</h2> ===
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<p>For this year’s project our team aimed at making the paper by using Agro bacterium.
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So, we confirmed whether carbohydrate developed from Agro bacterium could be used as the raw material for making paper.</p>
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<p></p>
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<p>Materials</p>
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<p>Agro bacterium tumefaciens C58　</p>
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<p>Agro bacterium tumefaciens C58　bacterial liquid</p>
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<p>  　　　　＋LB medium　　　　  　  2 ml</p>
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<p>  　　　　＋ampicillin　　　　　　        　2 µl</p>
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<p>  　　　　＋Agro bacterium tumefaciens C58</p>
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<p>Congo red</p>
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<p>LB medium</p>
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<p>　　　　＋1.0％(w/v)　 Triptone　　　　 2 g</p>
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<p>　　　　＋0.5％(w/v)　 yeast extract　  1 g</p>
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<p>　　　　＋1.0％(w/v)　　NaCl　　　　　 2 g</p>
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<p></p>
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<p>Sample 1 curdlan 15 µl　　　　  ＋Congo red 5 µl　＋Agro bacterium precipitationSample 1 curdlan 15 µl　　　　  ＋Congo red 5 µl　＋Agro bacterium precipitationSample 1 curdlan 15 µl　　　　  ＋Congo red 5 µl　＋Agro bacterium precipitation</p>
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<p>Sample 2 de-ionized water 15 µl　＋Congo red 5 µl　＋Agro bacterium precipitation</p>
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<p></p>
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<p>We centrifuged Sample 1 and 2 in 5 minutes with cooled centrifuges(4 ℃,13000 rpm).</p>
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<p>As a result, Sample 1 had red precipitates. Sample 2 rationally melted.</p>
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<p>So, we confirmed the presence of carbohydrate in the precipitates obtained from Agro bacterium.</p>
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<p></p>
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<p>Next, we found that carbohydrate could be produced when soy milk is added to the fluid bacteria</p>
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<p>So, we experimented for the confirmation of the above findings.</p>
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<p></p>
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<p>Sample 3　LB medium 7 ml ＋Agro bacterium＋soy 40 µl</p>
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<p>Sample 4　LB medium 7 ml ＋Agro bacterium</p>
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<p>Sample 5　LB medium 7 ml　　          　 ＋soy 40 µl</p>
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<p>Sample 6　LB medium 7 ml</p>
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<p></p>
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<p>We centrifuged Sample 1 and 2 in 5 minutes with cooled centrifuges(4 ℃,13000 rpm).</p>
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<p>As a result, Sample 1 had red precipitates. Sample 2 rationally melted.</p>
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<p>So, we confirmed the presence of carbohydrate in the precipitates obtained from Agro bacterium.</p>
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<p></p>
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<p>Sample 2 de-ionized water 15 µl　＋Congo red 5 µl　＋Agro bacterium precipitation</p>
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<p></p>
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<p>We centrifuged Sample 1 and 2 in 5 minutes with cooled centrifuges(4 ℃,13000 rpm).</p>
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<p>As a result, Sample 1 had red precipitates. Sample 2 rationally melted.</p>
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<p>So, we confirmed the presence of carbohydrate in the precipitates obtained from Agro bacterium.</p>
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<p></p>
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<p></p>
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<p>Next, we found that carbohydrate could be produced when soy milk is added to the fluid bacteria<p/>
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<p>So, we experimented for the confirmation of the above findings.</p>
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<p></p>
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<p>Sample 3　LB medium 7 ml ＋Agro bacterium＋soy 40 µl</p>
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<p>Sample 4　LB medium 7 ml ＋Agro bacterium</p>
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<p>Sample 5　LB medium 7 ml　　          　 ＋soy 40 µl</p>
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<p>Sample 6　LB medium 7 ml</p>
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<p></p>
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<p>We cultivated Sample 3 ~ 6 at 28 ℃.for 12 hours</p>
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<p>Then, Sample1 and 2 produced a white film like substance but Sample 3 and 4 did not.</p>
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<p>We put the white films from Sample 1 and 2 into micro-tubes.</p>
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<p></p>
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<p></p>
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<p></p>
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<p></p>
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<p>Sample 7　 supernatant(Sample 1) 15 µl　＋Congo red 5 µl</p>
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<p>Sample 8　 supernatant(Sample 1) 15 µl　＋Congo red 5 µl　＋de-ionized water 1 ml</p>
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<p>Sample 9　 supernatant(Sample 2) 15 µl　＋Congo red 5 µl　</p>
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<p>Sample 10　supernatant(Sample 2) 15 µl　＋Congo red 5 µl　＋de-ionized water 1 ml</p>
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<p>Sample 11　Congo red 5 µl　＋de-ionized water 1 ml</p>
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<p></p>
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<p>As a result, Sample 7 ~ 10 gave red precipitates but Sample 11 did not.</p>
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<p>So, we confirmed Sample 7 ~ 10 had carbohydrate.</p>
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<p></p>
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<p></p>
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<p></p>
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<p></p>
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<p>Through these experiments, we confirmed that Agro bacterium can discharge carbohydrate without soy milk.</p>
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<p></p>
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<p></p>
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<p>Reference</p>
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<p>1, Laboratory maintenance of Agrobacterium.</p>
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<p>(http://europepmc.org/articles/PMC3350319)</p>
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<p>2, Coordination of Division and Development Influences Complex Multicellular Behavior in Agrobacterium tumefaciens (Jinwoo Kim.¤, Jason E. Heindl., Clay Fuqua)</p>
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<p>3, Kirin Kyowa Foods Company (http://www.kirinkyowa-foods.co.jp/products/curdlan/)</p>
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<p>4, Howard Hughes Molecular Biology Summer Research Program Poster, Austin, TX , August, 1995 Microcopy of Curdlan  Structure</p>
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<p>5, Iain M. Cheeseman and R.Malcom Brown,Jr, Department of Botanty , The University of Texas at Austin,TX,78713</p>
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<p>(http://www.botanty.utexas.edu/facstaff/facpages/mbroun/ongres/icheeze.htm)</p>
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=== <h2>大瀧の実験</h2> ===
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=== <h2>カードラン</h2> ===
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=== <h2>セルC</h2> ===
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== <h2>CelC</h2> ==
<P></p>
<P></p>
<p>Agro Notebook</p>
<p>Agro Notebook</p>
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<p>CelC gene had produced clone from Agrobacterium tumefaciens C58, but I confirmed whether it’s true or not. CelC gene has restriction enzyme sites,EcoRI and BamHI. </p>
<p>CelC gene had produced clone from Agrobacterium tumefaciens C58, but I confirmed whether it’s true or not. CelC gene has restriction enzyme sites,EcoRI and BamHI. </p>
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{{Biwako_Nagahama/header}}
 
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=== <h2>Binary Vector</h2> ===
 
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<p>[[File:Biwakonagahama/pBI107_kei1.png]]</p>
 
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== <h2>E.coli-ink</h2> ==
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[[File:Biwako-Nagahama_IGEM＿訂正版図1.png|200px|left]]
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<div style>
 +
<table border="1">
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<p>
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<tr><td>No</td><td>Name</td><td>volume</td></tr>
 +
<tr><td>1</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>2</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>3</td><td>CelC_nonRE</td><td>12μL</td></tr>
 +
<tr><td>4</td><td>CelC/BamHI</td><td>12μL</td></tr>
 +
<tr><td>5</td><td>CelC/EcoRI</td><td>12μL</td></tr>
 +
<tr><td>6</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>7</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>8</td><td>-</td><td>-</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
<h3>Inverse PCR</h3>
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<div style>
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    <div class="spacer"></div>
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  </div>
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  <div class="spacer"></div>
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<p> Inverse PCR
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</p>
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 +
 
 +
<div style>
 +
<table border="1">
 +
<p>
 +
<tr><td>Name</td><td>volume</td></tr>
 +
<tr><td>10xEX Taq Buffer  </td><td>5μL</td></tr>
 +
<tr><td>dNTP Mixture(2.5mM)</td><td>4μL</td></tr>
 +
<tr><td>10pmol/µl  Primer F</td><td>2.5μL</td></tr>
 +
<tr><td>10pmol/µl  Primer R</td><td>2.5μL</td></tr>
 +
<tr><td>PCR反応液　No.9</td><td>1µl</td></tr>
 +
<tr><td>Ex Taq HS (5U/µl)</td><td>0.25µl</td></tr>
 +
<tr><td>dH2O      </td><td>34.75µl</td></tr>
 +
<tr><td>Total</td><td>50µl</td></tr>
 +
</p>
 +
</table>
</div>
</div>
 +
<h3>Inverse PCR</h3>
 +
<div style>
-
<div class="spacer"></div>
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 +
 
 +
 
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<p>※Restriction Enzyme sol.(line No.5 CelC_EcoRI)which has 1M NaCl  dissolved in it, so, the band in lane no.5 appeared as upper-side.
 +
</p>
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<p> CelC_Fw:  TGACGAAAGCACTGATCTGC
 +
 
 +
</p>
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<p> CelC_Rv:  GAAAAGATCGAAACGGTGG
 +
</p>
 +
 
 +
<div style>
 +
<table border="1">
 +
<p>
 +
<tr><td>94℃　　　2min</td><td></td></tr>
 +
<tr><td>↓</td><td></td></tr>
 +
<tr><td></td><td></td></tr>
 +
<tr><td>98℃　　　10sec</td><td></td></tr>
 +
<tr><td>49℃　　　30sec</td><td>35cycles</td>
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<tr><td>72℃　　　2min</td><td></td></tr>
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<tr><td> ↓</td><td></td></tr>
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<tr><td>72℃　　　2min</td><td></td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
<h3>Inverse PCR</h3>
 +
<div style>
 +
 
 +
 
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<p> TA cloning of CelC
 +
</p>
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 +
[[File:Biwako-Nagahama T.Ksenpai celC4.png|500px|]]
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 +
<p>16℃ 30min incubate
 +
</p>
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<p> Ligation of CelC/pMD20 and Transformation in JM109.
 +
</p>
 +
 
 +
[[File:Biwako-Nagahama_T.Ksenpai_celC5.png|500px|]]
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 +
 
 +
[[File:Biwako-Nagahama_T.Ksenpai_celC6.png|500px|]]
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<p> Cells were stored on ice for 30min.
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</p>
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<p> After 42℃ 30sec heat shock, cells were stored on ice for 2min.
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</p>
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<p> Then cells were pre-cultured at 37℃ for 1hr, plated to Ampicillin plate.
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</p>
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<p>6/1 Liquid clluture
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</p>
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<p> CelC/pMD20 22 samples at 37°C, for overnight.
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</p>
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<p>6/2 MiniPrep of CelC
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</p>
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 +
 
 +
<div style>
 +
<table border="1">
 +
<p><tr><td>No</td><td>Name</td><td>volume</td></tr>
 +
<tr><td>1</td><td>-</td><td>-</td></tr>
 +
<tr><td>2</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>3</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>4</td><td>Blue1</td><td>12μL</td></tr>
 +
<tr><td>5</td><td>Blue2</td><td>12μL</td></tr>
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<tr><td>6</td><td>Blue3</td><td>12μL</td></tr>
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<tr><td>7</td><td>Blue4</td><td>12μL</td></tr>
 +
<tr><td>8</td><td>Blue5</td><td>12μL</td></tr>
 +
<tr><td>9</td><td>Blue6</td><td>12μL</td></tr>
 +
<tr><td>10</td><td>White1</td><td>12μL</td></tr>
 +
<tr><td>11</td><td>White2</td><td>12μL</td></tr>
 +
<tr><td>12</td><td>White3</td><td>12μL</td></tr>
 +
<tr><td>13</td><td>White4</td><td>12μL</td></tr>
 +
<tr><td>14</td><td>White5</td><td>12μL</td></tr>
 +
<tr><td>15</td><td>λ-HindⅢ</td><td>10μL</td></tr>
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<tr><td>16</td><td>500bp DNA ladder</td><td>10μL</td></tr>
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<tr><td>17</td><td>-</td><td>-</td></tr></p>
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</table>
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</div>
 +
<div style>
 +
<table border="1">
 +
<p><tr><td>No</td><td>Name</td><td>volume</td></tr>
 +
<tr><td>1</td><td>-</td><td>-</td></tr>
 +
<tr><td>2</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>3</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>4</td><td>White6</td><td>12μL</td></tr>
 +
<tr><td>5</td><td>White7</td><td>12μL</td></tr>
 +
<tr><td>6</td><td>White8</td><td>12μL</td></tr>
 +
<tr><td>7</td><td>White9</td><td>12μL</td></tr>
 +
<tr><td>8</td><td>White10</td><td>12μL</td></tr>
 +
<tr><td>9</td><td>White11</td><td>12μL</td></tr>
 +
<tr><td>10</td><td>White12</td><td>12μL</td></tr>
 +
<tr><td>11</td><td>White13</td><td>12μL</td></tr>
 +
<tr><td>12</td><td>White14</td><td>12μL</td></tr>
 +
<tr><td>13</td><td>White15</td><td>12μL</td></tr>
 +
<tr><td>14</td><td>White16</td><td>12μL</td></tr>
 +
<tr><td>15</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>16</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>17</td><td>500bp DNA ladder</td><td>10μL</td></tr></p>
 +
</table>
 +
</div>
 +
 
 +
<h3>Inverse PCR</h3>
 +
<div style>
 +
 
 +
 
 +
 
 +
[[File:Biwako-Nagahama_T.Ksenpai_celC8.png|500px|]]
 +
 
 +
<p> Linear CelC/pMD20 DNA :2736bp. This CelC/pMD20 sample is cccDNA. So,white 6,white 7,white 8,white 9,white 14 probably picked up CelC/pMD20.
 +
</p>
 +
<p>6/3 Restriction Enzyme of CelC/pMD20
 +
</p>
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<p> CelC gene had produced clone from Agrobacterium tumefaciens C58, I confirmed whether it’s true or not. CelC/pMD20 gene has 2 restriction enzyme sites,BamHI
 +
</p>
 +
 
 +
[[File:Biwako-Nagahama_T.Ksenpai_celC9.png|500px|]]
 +
 
 +
 
 +
[[File:Biwako-Nagahama T.Ksenpai celC10.png|500px|]]
 +
 
 +
<p> I confirmed the direction of CelC gene.
 +
</p>
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<p>6/ Sequence of CelC/pMD20
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</p>
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 +
[[File:Biwako-Nagahama_T.Ksenpai_celC11.png|500px|]]
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<p>7/18 PointMutation of CelC
 +
</p>
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<p> CelC gene had Restriction Enzyme Site,EcoRI. We directed the EcoRI site.
 +
</p>
 +
 
 +
[[File:Biwako-Nagahama_T.Ksenpai_celC12.png|400px|left]]
 +
 
 +
<div style>
 +
<table border="1">
 +
<p><tr><td>No</td><td>Name</td><td>volume</td></tr>
 +
<tr><td>1</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>2</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>3</td><td>CelC Negative Front Fragment</td><td>12μL</td></tr>
 +
<tr><td>4</td><td>CelC Negative Rear Fragment</td><td>12μL</td></tr>
 +
<tr><td>5</td><td>CelC No.6 Front Fragment</td><td>12μL</td></tr>
 +
<tr><td>6</td><td>CelC No.6 Rear Fragment</td><td>12μL</td></tr>
 +
<tr><td>7</td><td>CelC No.7 Front Fragment</td><td>12μL</td></tr>
 +
<tr><td>8</td><td>CelC No.7 Rear Fragment</td><td>12μL</td></tr>
 +
<tr><td>9</td><td>CelC No.8 Front Fragment</td><td>12μL</td></tr>
 +
<tr><td>10</td><td>CelC No.8 Rear Fragment</td><td>12μL</td></tr>
 +
<tr><td>11</td><td>CelC No.9 Front Fragment</td><td>12μL</td></tr>
 +
<tr><td>12</td><td>CelC No.9 Rear Fragment</td><td>12μL</td></tr>
 +
<tr><td>13</td><td>CelC No.14 Front Fragment</td><td>12μL</td></tr>
 +
<tr><td>14</td><td>CelC No.14 Rear Fragment</td><td>12μL</td></tr>
 +
<tr><td>15</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>16</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
 
 +
 
 +
<p> Front
 +
</p>
 +
<p> Fw1 Primer:
 +
</p>
 +
<p> TATATATTCTAGATGAAGAGCGGGATTTCG
 +
 
 +
</p>
 +
<p> Rv2 Primer:
 +
</p>
 +
<p> CATTATATCCGAACTCCGGCTG
 +
</p>
 +
<p> Rear
 +
</p>
 +
<p> Fw2 Primer:
 +
</p>
 +
<p> AGCCGGAGTTCGGATATAATGC
 +
</p>
 +
 
 +
 
 +
<p> Rv1 Primer:
 +
</p>
 +
<p> CAGCACGAACTAGTATTATTATCATCGGC
 +
</p>
 +
 
 +
<div style>
 +
<table border="1">
 +
<p>
 +
<tr><td>5×PS buffer</td><td>10μL</td></tr>
 +
<tr><td>dNTP Mixture(2.5mM)</td><td>4μL</td></tr>
 +
<tr><td>10pmol/μL Primer Fw1 or Fw2</td><td>1μL</td></tr>
 +
<tr><td>10pmol/μL Primer Rv1 or Rv2</td><td>1μL</td></tr>
 +
<tr><td>Template</td><td>1μL</td></tr>
 +
<tr><td>Prime STAR HS(5U/μL)</td><td>0.5μL</td></tr>
 +
<tr><td>dH2O</td><td>32.5μL</td></tr>
 +
<tr><td>Total</td><td>50μL</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
 
 +
 
 +
 
 +
<p>7/19 Gel Purification of CelC gene’s Front Fragment and Rear Fragment
 +
</p>
 +
<p> Front Fragment DNA and Rear Fragment DNA that 765bp and 347bp band performed Gel Purification by illustra GFX PCR Purification Kit.
 +
</p>
 +
<p>7/21 CelC gene’s Front Fragment and Rear Fragment Overlap PCR
 +
 
 +
</p>
 +
 
 +
[[File:Biwako-Nagahama_T.Ksenpai_celC16.png|200px|left]]
 +
 
 +
<div style>
 +
<table border="1">
 +
<p><tr><td>No</td><td>Name</td><td>volume</td></tr>
 +
<tr><td>1</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>2</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>3</td><td>CelC N Assembly DNA</td><td>12μL</td></tr>
 +
<tr><td>4</td><td>CelC No.6 Assembly DNA</td><td>12μL</td></tr>
 +
<tr><td>5</td><td>CelC No.7 Assembly DNA</td><td>12μL</td></tr>
 +
<tr><td>6</td><td>CelC No.8 Assembly DNA</td><td>12μL</td></tr>
 +
<tr><td>7</td><td>CelC No.9 Assembly DNA</td><td>12μL</td></tr>
 +
<tr><td>8</td><td>CelC No.14 Assembly DNA</td><td>12μL</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
 
 +
 
 +
 
 +
<p> No.6,7,8,9,14 each fragment Overlap PCR completed.
 +
 
 +
</p>
 +
<p> I selected No.8.
 +
 
 +
</p>
 +
<p>7/22 Gel Purification of CelC gene No.8
 +
 
 +
</p>
 +
<p> No.8 DNA that about 1ooo bp band performed Gel Purification by illustra GFX PCR Purification Kit.
 +
 
 +
</p>
 +
<p>8/1 Adapter PCR of CelC gene No.8 (BioBrick Part)
 +
 
 +
</p>
 +
 
 +
 
 +
[[File:Biwako-Nagahama_T.Ksenpai_celC18.png|200px|left]]
 +
 
 +
 
 +
<div style>
 +
<table border="1">
 +
<p><tr><td>No</td><td>Name</td><td>volume</td></tr>
 +
<tr><td>1</td><td>2-log DNA ladder</td><td>5μL</td></tr>
 +
<tr><td>2</td><td>λ-HindⅢ</td><td>6μL</td></tr>
 +
<tr><td>3</td><td>PCR sample</td><td>18μL</td></tr>
 +
<tr><td>4</td><td>-</td><td>-</td></tr>
 +
<tr><td>5</td><td>-</td><td>-</td></tr>
 +
<tr><td>6</td><td>-</td><td>-</td></tr>
 +
<tr><td>7</td><td>-</td><td>-</td></tr>
 +
<tr><td>8</td><td>-</td><td>-</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
 
 +
<p> Fw Primer:GTTTCTTCGAATTCGCGGCCGCTTCTAGATG
 +
</p>
 +
<p> Rv Primer:GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATC
 +
</p>
 +
<p>8/11 BioBrick of CelC Restrict Enzyme ,EcoRI.
 +
 
 +
</p>
 +
 
 +
[[File:Biwako-Nagahama_T.Ksenpai_celC20.png|200px|left]]
 +
 
 +
<div style>
 +
<table border="1">
 +
<p><tr><td>No</td><td>Name</td><td>volume</td></tr>
 +
<tr><td>1</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>2</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>3</td><td>PCR sample A non RE</td><td>12μL</td></tr>
 +
<tr><td>4</td><td>PCR sample A EcoRⅠ RE</td><td>12μL</td></tr>
 +
<tr><td>5</td><td>PCR sample B non RE</td><td>12μL</td></tr>
 +
<tr><td>6</td><td>PCR sample B EcoRⅠ RE</td><td>12μL</td></tr>
 +
<tr><td>7</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>8</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
 
 +
 
 +
 
 +
<p> I confirmed BioBrick of CelC non-Restriction Enzyme ,EcoRI.
 +
</p>
 +
<p>9/5 Brick Part of CelC and pSB1C3 Restrict Enzyme,EcoRI and PstI.
 +
 
 +
</p>
 +
 
 +
<div style>
 +
<table border="1">
 +
<p>
 +
<tr><td>・CelC</td><td></td></tr>
 +
<tr><td>S.D.W</td><td>10μL</td></tr>
 +
<tr><td>EcoRⅠ</td><td>1μL</td></tr>
 +
<tr><td>PstⅠ</td><td>1μL</td></tr>
 +
<tr><td>PCR sample</td><td>6μL</td></tr>
 +
<tr><td>10×H buffer</td><td>2μL</td></tr>
 +
<tr><td>Total</td><td>20μL</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
<div style>
 +
<table border="1">
 +
<p>
 +
<tr><td>・pSB1C3</td><td></td></tr>
 +
<tr><td>S.D.W</td><td>10μL</td></tr>
 +
<tr><td>EcoRⅠ</td><td>1μL</td></tr>
 +
<tr><td>PstⅠ</td><td>1μL</td></tr>
 +
<tr><td>pSB1C3</td><td>6μL</td></tr>
 +
<tr><td>10×H buffer</td><td>2μL</td></tr>
 +
<tr><td>Total</td><td>20μL</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
 
 +
 
 +
 
 +
<p>9/5 Ligation of CelC/pSB1C3 and Transformation in JM109.
 +
 
 +
</p>
 +
 
 +
<div style>
 +
<table border="1">
 +
<p>
 +
<tr><td>CelC/EcoRⅠ,PstⅠ(100ng)</td><td>1μL</td></tr>
 +
<tr><td>pSB1C3/EcoRⅠ,PstⅠ(50ng)</td><td>2μL</td></tr>
 +
<tr><td>Ligation kit ver.2</td><td>3μL</td></tr>
 +
<tr><td>Total</td><td>6μL</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
 
 +
 
 +
<p>9/7 Colony PCR of CelC/pSB1C3
 +
 
 +
</p>
 +
 
 +
[[File:Biwako-Nagahama_T.Ksenpai_celC24.png|450px|left]]
 +
 
 +
<div style>
 +
<table border="1">
 +
<p><tr><td>No</td><td>Name</td><td>volume</td></tr>
 +
<tr><td>1</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>2</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>3</td><td>No.1</td><td>12μL</td></tr>
 +
<tr><td>4</td><td>No.2</td><td>12μL</td></tr>
 +
<tr><td>5</td><td>No.3</td><td>12μL</td></tr>
 +
<tr><td>6</td><td>No.4</td><td>12μL</td></tr>
 +
<tr><td>7</td><td>No.5</td><td>12μL</td></tr>
 +
<tr><td>8</td><td>No.6</td><td>12μL</td></tr>
 +
<tr><td>9</td><td>No.7</td><td>12μL</td></tr>
 +
<tr><td>10</td><td>No.8</td><td>12μL</td></tr>
 +
<tr><td>11</td><td>No.9</td><td>12μL</td></tr>
 +
<tr><td>12</td><td>No.10</td><td>12μL</td></tr>
 +
<tr><td>13</td><td>No.11</td><td>12μL</td></tr>
 +
<tr><td>14</td><td>No.12</td><td>12μL</td></tr>
 +
<tr><td>15</td><td>No.13</td><td>12μL</td></tr>
 +
<tr><td>16</td><td>No.14</td><td>12μL</td></tr>
 +
<tr><td>17</td><td>No.15</td><td>12μL</td></tr>
 +
<tr><td>18</td><td>-</td><td>-</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
 
 +
 
 +
<p>CelC/pSB1C3 DNA(VR-VF2) :1370bp. So,No.5 and No.12 probably picked up CelC/pSB1C3
 +
</p>
 +
<p>9/8 Miniprep of CelC/pSB1C3
 +
 
 +
</p>
 +
 
 +
[[File:Biwako-Nagahama_T.Ksenpai_celC27.png|450px|left]]
 +
 
 +
<div style>
 +
<table border="1">
 +
<p><tr><td>No</td><td>Name</td><td>volume</td></tr>
 +
<tr><td>1</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>2</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>3</td><td>No.1</td><td>12μL</td></tr>
 +
<tr><td>4</td><td>No.2</td><td>12μL</td></tr>
 +
<tr><td>5</td><td>No.3</td><td>12μL</td></tr>
 +
<tr><td>6</td><td>No.4</td><td>12μL</td></tr>
 +
<tr><td>7</td><td>No.5</td><td>12μL</td></tr>
 +
<tr><td>8</td><td>No.6</td><td>12μL</td></tr>
 +
<tr><td>9</td><td>No.7</td><td>12μL</td></tr>
 +
<tr><td>10</td><td>No.8</td><td>12μL</td></tr>
 +
<tr><td>11</td><td>No.9</td><td>12μL</td></tr>
 +
<tr><td>12</td><td>No.10</td><td>12μL</td></tr>
 +
<tr><td>13</td><td>No.11</td><td>12μL</td></tr>
 +
<tr><td>14</td><td>No.12</td><td>12μL</td></tr>
 +
<tr><td>15</td><td>No.13</td><td>12μL</td></tr>
 +
<tr><td>16</td><td>No.14</td><td>12μL</td></tr>
 +
<tr><td>17</td><td>No.15</td><td>12μL</td></tr>
 +
<tr><td>18</td><td>-</td><td>-</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
 
 +
 
 +
 
 +
<p> Linear CelC/pSB1C3 DNA :3126bp. This CelC/pSB1C3 sample is cccDNA. So,No.5 and No.12 probably picked up CelC/pSB1C3.
 +
 
 +
 
 +
</p>
 +
<p>9/15. Sequence of CelC Brick Part sequence.
 +
 
 +
</p>
 +
<p> Result of NCBI BLAST
 +
</p>
 +
 
 +
 
 +
[[File:Biwako-Nagahama_T.Ksenpai_celC28.png|500px]]
 +
 
 +
 
 +
=== <h2> CrdS</h2> ===
 +
<h3>Cloning of CrdS and Restriction Enzyme</h3>
 +
<h5>By Koki Tsutsumi</h5>
 +
<p>CrdS gene had produced clone from Agrobacterium tumefaciens C58, but I confirmed whether it’s true or not. CrdS gene has restriction enzyme sites,EcoRI and PstI. </p>
 +
[[File:Biwako-Nagahama_E.P_tutumi1.png|left|170px]]
 +
<div style>
 +
<table border="1">
 +
<p>
 +
<tr><td>No</td><td>Name</td><td>volume</td></tr>
 +
<tr><td>1</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>2</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>3</td><td>CrdS</td><td>12μL</td></tr>
 +
<tr><td>4</td><td>-</td><td>-</td></tr>
 +
<tr><td>5</td><td>-</td><td>-</td></tr>
 +
<tr><td>6</td><td>-</td><td>-</td></tr>
 +
<tr><td>7</td><td>-</td><td>-</td></tr>
 +
<tr><td>8</td><td>-</td><td>-</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
[[File:Biwako-Nagahama_E.P_tutumi2.png|left|170px]]
 +
<div style>
 +
<table border="1">
 +
<p>
 +
<tr><td>No</td><td>Name</td><td>volume</td></tr>
 +
<tr><td>1</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>2</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>3</td><td>CrdS_nonRE</td><td>12μL</td></tr>
 +
<tr><td>4</td><td>CrdS/EcoRⅠ</td><td>12μL</td></tr>
 +
<tr><td>5</td><td>CrdS/PstⅠ</td><td>12μL</td></tr>
 +
<tr><td>6</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>7</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>8</td><td>-</td><td>-</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
<h3>Inverse PCR</h3>
 +
<div style>
 +
<table border="1">
 +
<p>
 +
<tr><td>Prime STAR MAX</td><td>25μL</td></tr>
 +
<tr><td>10pmol/μL Primer F</td><td>1μL</td></tr>
 +
<tr><td>10pmol/μL Primer R</td><td>1μL</td></tr>
 +
<tr><td>PCR反応液(2ng)</td><td>1μL</td></tr>
 +
<tr><td>dH2O</td><td>22μL</td></tr>
 +
<tr><td>Total</td><td>22μL</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
<p>CrdS_Fw:  AGTACGATCCGCTATTTTCCCG</p>
 +
<p>CrdS_Rv:  CAGACCAAGATTTCGCGAACTC</p>
 +
----
 +
<p>94℃ 2min</p>
 +
<p>↓</p>
 +
<p>98℃ 10sec</p>
 +
<p>48℃ 30sec&nbsp;&nbsp;&nbsp;&nbsp;35cycles</p>
 +
<p>72℃ 2min</p>
 +
<p>↓</p>
 +
<p>72℃ 2min</p>
 +
----
 +
<h3>TA cloning of CrdS</h3>
 +
<div style>
 +
<table border="1">
 +
<tr><td>PCR product</td><td>1μL</td></tr>
 +
<tr><td>pMD20</td><td>2μL</td></tr>
 +
<tr><td>Ligation kit ver.2</td><td>3μL</td></tr>
 +
</table>
 +
</div>
 +
16℃ 30min incubate
 +
<h3>Ligation of CrdS/pMD20 and Transformation in JM109</h3>
 +
<div style>
 +
<table border="1">
 +
<tr><td>Competent cell</td><td>100μL</td></tr>
 +
<tr><td>DNA</td><td>6μL</td></tr>
 +
</table>
 +
</div>
 +
<p>↓Cells were stored on ice for 30min. </p>
 +
<p>↓After 42℃ 30sec heat shock, cells were stored on ice for 2min.</p>
 +
<p>↓Then cells were pre-cultured at 37℃ for 1hr, plated to Ampicillin plate. </p>
 +
<h3>Liquid culluture</h3>
 +
CrdS/pMD20 21 samples at 37℃,for overnight
 +
<h3>MiniPrep of CrdS/pMD20</h3>
 +
Linear CrdS/pMD20 DNA :4995bp. This CrdS/pMD20 sample is cccDNA. So, probably picked up CrdS/pMD20.
 +
[[File:Biwako-Nagahama_E.P_tutumi3.png|left|170px]]
 +
<div style>
 +
<table border="1">
 +
<p>
 +
<tr><td>No</td><td>Name</td><td>volume</td></tr>
 +
<tr><td>1</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>2</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>3</td><td>CrdS No.5</td><td>12μL</td></tr>
 +
<tr><td>4</td><td>CrdS No.6</td><td>12μL</td></tr>
 +
<tr><td>5</td><td>CrdS No.8</td><td>12μL</td></tr>
 +
<tr><td>6</td><td>CrdS No.15</td><td>12μL</td></tr>
 +
<tr><td>7</td><td>CrdS No.17</td><td>12μL</td></tr>
 +
<tr><td>8</td><td>-</td><td>-</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
<h3>Restriction Enzyme of CrdS/pMD20</h3>
 +
CrdS gene had produced clone from Agrobacterium tumefaciens C58, I confirmed whether it’s true or not. CrdS/pMD20 gene has 2 restriction enzyme sites,EcoRI.
 +
[[File:Biwako-Nagahama_E.P_tutumi4.png|left|170px]]
 +
<div style>
 +
<table border="1">
 +
<p>
 +
<tr><td>No</td><td>Name</td><td>volume</td></tr>
 +
<tr><td>1</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>2</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>3</td><td>CrdS No.5</td><td>12μL</td></tr>
 +
<tr><td>4</td><td>CrdS No.6</td><td>12μL</td></tr>
 +
<tr><td>5</td><td>CrdS No.2</td><td>12μL</td></tr>
 +
<tr><td>6</td><td>CrdS No.15</td><td>12μL</td></tr>
 +
<tr><td>7</td><td>CrdS No.17</td><td>12μL</td></tr>
 +
<tr><td>8</td><td>-</td><td>-</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
I comfirmed the direction of CrdS gene.
 +
<h3>Sequence of CrdS/pMD20</h3>
 +
[[File:Biwako-Nagahama_Sequences_tutumi5.png|700px|]]
 +
<h3>PointMutation of CrdS</h3>
 +
CrdS gene had Restriction Enzyme Site,EcoRI. We removed the EcoRI site.
 +
[[File:Biwako-Nagahama_E.P_tutumi6.png|left|]]
 +
<div style>
 +
<table border="1">
 +
<p>
 +
<tr><td>No</td><td>Name</td><td>volume</td></tr>
 +
<tr><td>1</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>2</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>3</td><td>CrdS Negative Front Fragment</td><td>12μL</td></tr>
 +
<tr><td>4</td><td>CrdS Negative Rear Fragment</td><td>12μL</td></tr>
 +
<tr><td>5</td><td>CrdS No.6 Front Fragment</td><td>12μL</td></tr>
 +
<tr><td>6</td><td>CrdS No.6 Middle Fragment</td><td>12μL</td></tr>
 +
<tr><td>7</td><td>CrdS No.6 Rear Fragment</td><td>12μL</td></tr>
 +
<tr><td>8</td><td>CrdS No.15 Front Fragment</td><td>12μL</td></tr>
 +
<tr><td>9</td><td>CrdS No.15 Middle Fragment</td><td>12μL</td></tr>
 +
<tr><td>10</td><td>CrdS No.15 Rear Fragment</td><td>12μL</td></tr>
 +
<tr><td>11</td><td>-</td><td>-</td></tr>
 +
<tr><td>12</td><td>-</td><td>-</td></tr>
 +
<tr><td>13</td><td>-</td><td>-</td></tr>
 +
<tr><td>14</td><td>-</td><td>-</td></tr>
 +
<tr><td>15</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>16</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
<div style='width:560;font:bold 14;color:black;text-align:left;>
 +
<p>Front</p>
 +
<p>Fw0 Primer:GGCCGCTTCTAGATGTATTTCAGTGC</p>
 +
<p>Rv1 Primer:CGTTTCGAGGGAGAACTCCAGCG</p>
 +
<p>Middle</p>
 +
<p>Fw1Primer:CGCTGGAGTTCTCCCTCGAAACG</p>
 +
<p>Rv2Primer:CAGCTCATCGGCCTGAAGCGCC</p>
 +
<p>Rear</p>
 +
<p>Fw2 Primer:GGCGCTTCAGGCCGATGAGCTG</p>
 +
<p>Rv0 Primer:GGCGCTACTAGTATTATTATCACCCGAATG</p>
 +
</div>
 +
<div align="left">
 +
<table border="1">
 +
<p>
 +
<tr><td>5xPS Buffer</td><td>10μL</td></tr>
 +
<tr><td>dNTP Mixture</td><td>4μL</td></tr>
 +
<tr><td>10pmol/µl  Primer Fw1 or Fw2</td><td>1μL</td></tr>
 +
<tr><td>10pmol/µl  Primer Rv2 or Rv1</td><td>1μL</td></tr>
 +
<tr><td>Templete</td><td>1μL</td></tr>
 +
<tr><td>Prime STAR HS</td><td>0.5μL</td></tr>
 +
<tr><td>dH2O</td><td>32.5μL</td></tr>
 +
<tr><td>Total</td><td>50μL</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
----
 +
<p>94℃ 2min</p>
 +
<p>↓</p>
 +
<p>98℃ 10sec</p>
 +
<p>55℃ 5sec&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;35cycles</p>
 +
<p>72℃ 70sec</p>
 +
<p>↓</p>
 +
<p>4℃ ∞</p>
 +
----
 +
<h3>Gel Purification of CrdS gene’s Front Fragment and Rear Fragment</h3>
 +
Front Fragment DNA and Rear Fragment DNA ,Middle Fragment DNA that 223 bp and 1037 bp ,782 bp band performed Gel Purification by illustra GFX PCR Purification Kit.
 +
<h3>CrdS gene’s Front Fragment and Rear Fragment Overlap PCR</h3>
 +
[[File:Biwako-Nagahama_E.P_tutumi7.png|left|170px]]
 +
<div style>
 +
<table border="1">
 +
<p>
 +
<tr><td>No</td><td>Name</td><td>volume</td></tr>
 +
<tr><td>1</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>2</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>3</td><td>CrdS N Front+Middle DNA</td><td>12μL</td></tr>
 +
<tr><td>4</td><td>CrdS No.6 Front+Middle DNA</td><td>12μL</td></tr>
 +
<tr><td>5</td><td>CrdS No.15 Front+Middle DNA</td><td>12μL</td></tr>
 +
<tr><td>6</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>7</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>8</td><td>-</td><td>-</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
[[File:Biwako-Nagahama_E.P_tutumi8.png|left|170px]]
 +
<div style>
 +
<table border="1">
 +
<p>
 +
<tr><td>No</td><td>Name</td><td>volume</td></tr>
 +
<tr><td>1</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>2</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>3</td><td>CrdS N Front+Rear DNA</td><td>12μL</td></tr>
 +
<tr><td>4</td><td>CrdS No.6 Front+Rear DNA</td><td>12μL</td></tr>
 +
<tr><td>5</td><td>CrdS No.15 Front+Rear DNA</td><td>12μL</td></tr>
 +
<tr><td>6</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>7</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>8</td><td>-</td><td>-</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
N, No.6, No.15 each fragment Overlap PCR completed.
 +
<h3>Gel Purification of CrdS</h3>
 +
No.8 DNA that about 2ooo bp band performed Gel Purification by illustra GFX PCR Purification Kit.
 +
<h3>Adapter PCR of CrdS gene (BioBrick Part)</h3>
 +
[[File:Biwako-Nagahama_E.P_tutumi9.png|left|170px]]
 +
<div style>
 +
<table border="1">
 +
<p>
 +
<tr><td>No</td><td>Name</td><td>volume</td></tr>
 +
<tr><td>1</td><td>2-log DNA ladder</td><td>5μL</td></tr>
 +
<tr><td>2</td><td>λ-HindⅢ</td><td>6μL</td></tr>
 +
<tr><td>3</td><td>PCR saample</td><td>18μL</td></tr>
 +
<tr><td>4</td><td>-</td><td>-</td></tr>
 +
<tr><td>5</td><td>-</td><td>-</td></tr>
 +
<tr><td>6</td><td>-</td><td>-</td></tr>
 +
<tr><td>7</td><td>-</td><td>-</td></tr>
 +
<tr><td>8</td><td>-</td><td>-</td></tr>
 +
</p>
 +
</table>
 +
</div>
 +
<p>Fw Primer:
 +
GTTTCTTCGAATTCGCGGCCGCTTCTAGATG</p>
 +
<p>Rv Primer:
 +
GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATC</p>
 +
<h3>Brick Part of CrdS Restrict Enzyme,EcoRI and PstI.</h3>
 +
[[File:Biwako-Nagahama_E.P_tutumi10.png|left|450px]]
 +
<div style>
 +
<table border="1">
 +
<p><tr><td>No</td><td>Name</td><td>volume</td></tr>
 +
<tr><td>1</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>2</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>3</td><td>CrdS N F+M non-RE</td><td>12μL</td></tr>
 +
<tr><td>4</td><td>CrdS N F+M EcoRⅠ-RE</td><td>12μL</td></tr>
 +
<tr><td>5</td><td>CrdS No.6 F+M non-RE</td><td>12μL</td></tr>
 +
<tr><td>6</td><td>CrdS No.6 F+M EcoRⅠ-RE</td><td>12μL</td></tr>
 +
<tr><td>7</td><td>CrdS No.15 F+M non-RE</td><td>12μL</td></tr>
 +
<tr><td>8</td><td>CrdS No.15 F+M EcoRⅠ</td><td>12μL</td></tr>
 +
<tr><td>9</td><td>CrdS N F+M+R non-RE</td><td>12μL</td></tr>
 +
<tr><td>10</td><td>CrdS N F+M+R PstⅠ-RE</td><td>12μL</td></tr>
 +
<tr><td>11</td><td>CrdS No.6  F+M+R non-RE</td><td>12μL</td></tr>
 +
<tr><td>12</td><td>Crds No.6  F+M+R PstI RE</td><td>12μL</td></tr>
 +
<tr><td>13</td><td>CrdS No.15  F+M+R non-RE</td><td>12μL</td></tr>
 +
<tr><td>14</td><td>CrdS No.15  F+M+R PstI RE</td><td>12μL</td></tr>
 +
<tr><td>15</td><td>λ-HindⅢ</td><td>10μL</td></tr>
 +
<tr><td>16</td><td>500bp DNA ladder</td><td>10μL</td></tr>
 +
<tr><td>17</td><td>-</td><td>-</td></tr>
 +
<tr><td>18</td><td>-</td><td>-</td></tr>
 +
</p>
 +
</table>
</div>
</div>
 +
N,No.6,No.15 of CrdS was cut on EcoRI site. Point Mutation of CrdS is unsuccessful.

Latest revision as of 02:31, 28 September 2013

Contents

Material & Method

CelC

Agro Notebook

5/31 Cloning of CelC and Restriction Enzyme

By Koki Tsutsumi

CelC gene had produced clone from Agrobacterium tumefaciens C58, but I confirmed whether it’s true or not. CelC gene has restriction enzyme sites,EcoRI and BamHI.


Biwako-Nagahama IGEM＿訂正版図1.png


NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3CelC_nonRE12μL
4CelC/BamHI12μL
5CelC/EcoRI12μL
6λ-HindⅢ10μL
7500bp DNA ladder10μL
8--

Inverse PCR



Inverse PCR


Namevolume
10xEX Taq Buffer 5μL
dNTP Mixture(2.5mM)4μL
10pmol/µl Primer F2.5μL
10pmol/µl Primer R2.5μL
PCR反応液　No.91µl
Ex Taq HS (5U/µl)0.25µl
dH2O 34.75µl
Total50µl

Inverse PCR



※Restriction Enzyme sol.(line No.5 CelC_EcoRI)which has 1M NaCl dissolved in it, so, the band in lane no.5 appeared as upper-side.

CelC_Fw: TGACGAAAGCACTGATCTGC

CelC_Rv: GAAAAGATCGAAACGGTGG

94℃　　　2min
↓
98℃　　　10sec
49℃　　　30sec35cycles
72℃　　　2min
↓
72℃　　　2min

Inverse PCR


TA cloning of CelC

Biwako-Nagahama T.Ksenpai celC4.png

16℃ 30min incubate

Ligation of CelC/pMD20 and Transformation in JM109.

Biwako-Nagahama T.Ksenpai celC5.png


Biwako-Nagahama T.Ksenpai celC6.png

Cells were stored on ice for 30min.

After 42℃ 30sec heat shock, cells were stored on ice for 2min.

Then cells were pre-cultured at 37℃ for 1hr, plated to Ampicillin plate.

6/1 Liquid clluture

CelC/pMD20 22 samples at 37°C, for overnight.

6/2 MiniPrep of CelC


NoNamevolume
1--
2500bp DNA ladder10μL
3λ-HindⅢ10μL
4Blue112μL
5Blue212μL
6Blue312μL
7Blue412μL
8Blue512μL
9Blue612μL
10White112μL
11White212μL
12White312μL
13White412μL
14White512μL
15λ-HindⅢ10μL
16500bp DNA ladder10μL
17--

NoNamevolume
1--
2500bp DNA ladder10μL
3λ-HindⅢ10μL
4White612μL
5White712μL
6White812μL
7White912μL
8White1012μL
9White1112μL
10White1212μL
11White1312μL
12White1412μL
13White1512μL
14White1612μL
15λ-HindⅢ10μL
16500bp DNA ladder10μL
17500bp DNA ladder10μL

Inverse PCR


Biwako-Nagahama T.Ksenpai celC8.png

Linear CelC/pMD20 DNA :2736bp. This CelC/pMD20 sample is cccDNA. So,white 6,white 7,white 8,white 9,white 14 probably picked up CelC/pMD20.

6/3 Restriction Enzyme of CelC/pMD20

CelC gene had produced clone from Agrobacterium tumefaciens C58, I confirmed whether it’s true or not. CelC/pMD20 gene has 2 restriction enzyme sites,BamHI

Biwako-Nagahama T.Ksenpai celC9.png


Biwako-Nagahama T.Ksenpai celC10.png

I confirmed the direction of CelC gene.

6/ Sequence of CelC/pMD20

Biwako-Nagahama T.Ksenpai celC11.png

7/18 PointMutation of CelC

CelC gene had Restriction Enzyme Site,EcoRI. We directed the EcoRI site.

Biwako-Nagahama T.Ksenpai celC12.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3CelC Negative Front Fragment12μL
4CelC Negative Rear Fragment12μL
5CelC No.6 Front Fragment12μL
6CelC No.6 Rear Fragment12μL
7CelC No.7 Front Fragment12μL
8CelC No.7 Rear Fragment12μL
9CelC No.8 Front Fragment12μL
10CelC No.8 Rear Fragment12μL
11CelC No.9 Front Fragment12μL
12CelC No.9 Rear Fragment12μL
13CelC No.14 Front Fragment12μL
14CelC No.14 Rear Fragment12μL
15λ-HindⅢ10μL
16500bp DNA ladder10μL


Front

Fw1 Primer:

TATATATTCTAGATGAAGAGCGGGATTTCG

Rv2 Primer:

CATTATATCCGAACTCCGGCTG

Rear

Fw2 Primer:

AGCCGGAGTTCGGATATAATGC


Rv1 Primer:

CAGCACGAACTAGTATTATTATCATCGGC

5×PS buffer10μL
dNTP Mixture(2.5mM)4μL
10pmol/μL Primer Fw1 or Fw21μL
10pmol/μL Primer Rv1 or Rv21μL
Template1μL
Prime STAR HS(5U/μL)0.5μL
dH2O32.5μL
Total50μL


7/19 Gel Purification of CelC gene’s Front Fragment and Rear Fragment

Front Fragment DNA and Rear Fragment DNA that 765bp and 347bp band performed Gel Purification by illustra GFX PCR Purification Kit.

7/21 CelC gene’s Front Fragment and Rear Fragment Overlap PCR

Biwako-Nagahama T.Ksenpai celC16.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3CelC N Assembly DNA12μL
4CelC No.6 Assembly DNA12μL
5CelC No.7 Assembly DNA12μL
6CelC No.8 Assembly DNA12μL
7CelC No.9 Assembly DNA12μL
8CelC No.14 Assembly DNA12μL


No.6,7,8,9,14 each fragment Overlap PCR completed.

I selected No.8.

7/22 Gel Purification of CelC gene No.8

No.8 DNA that about 1ooo bp band performed Gel Purification by illustra GFX PCR Purification Kit.

8/1 Adapter PCR of CelC gene No.8 (BioBrick Part)


Biwako-Nagahama T.Ksenpai celC18.png


NoNamevolume
12-log DNA ladder5μL
2λ-HindⅢ6μL
3PCR sample18μL
4--
5--
6--
7--
8--

Fw Primer:GTTTCTTCGAATTCGCGGCCGCTTCTAGATG

Rv Primer:GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATC

8/11 BioBrick of CelC Restrict Enzyme ,EcoRI.

Biwako-Nagahama T.Ksenpai celC20.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3PCR sample A non RE12μL
4PCR sample A EcoRⅠ RE12μL
5PCR sample B non RE12μL
6PCR sample B EcoRⅠ RE12μL
7λ-HindⅢ10μL
8500bp DNA ladder10μL


I confirmed BioBrick of CelC non-Restriction Enzyme ,EcoRI.

9/5 Brick Part of CelC and pSB1C3 Restrict Enzyme,EcoRI and PstI.

・CelC
S.D.W10μL
EcoRⅠ1μL
PstⅠ1μL
PCR sample6μL
10×H buffer2μL
Total20μL

・pSB1C3
S.D.W10μL
EcoRⅠ1μL
PstⅠ1μL
pSB1C36μL
10×H buffer2μL
Total20μL


9/5 Ligation of CelC/pSB1C3 and Transformation in JM109.

CelC/EcoRⅠ,PstⅠ(100ng)1μL
pSB1C3/EcoRⅠ,PstⅠ(50ng)2μL
Ligation kit ver.23μL
Total6μL


9/7 Colony PCR of CelC/pSB1C3

Biwako-Nagahama T.Ksenpai celC24.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3No.112μL
4No.212μL
5No.312μL
6No.412μL
7No.512μL
8No.612μL
9No.712μL
10No.812μL
11No.912μL
12No.1012μL
13No.1112μL
14No.1212μL
15No.1312μL
16No.1412μL
17No.1512μL
18--


CelC/pSB1C3 DNA(VR-VF2) :1370bp. So,No.5 and No.12 probably picked up CelC/pSB1C3

9/8 Miniprep of CelC/pSB1C3

Biwako-Nagahama T.Ksenpai celC27.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3No.112μL
4No.212μL
5No.312μL
6No.412μL
7No.512μL
8No.612μL
9No.712μL
10No.812μL
11No.912μL
12No.1012μL
13No.1112μL
14No.1212μL
15No.1312μL
16No.1412μL
17No.1512μL
18--


Linear CelC/pSB1C3 DNA :3126bp. This CelC/pSB1C3 sample is cccDNA. So,No.5 and No.12 probably picked up CelC/pSB1C3.

9/15. Sequence of CelC Brick Part sequence.

Result of NCBI BLAST


Biwako-Nagahama T.Ksenpai celC28.png


CrdS

Cloning of CrdS and Restriction Enzyme

By Koki Tsutsumi

CrdS gene had produced clone from Agrobacterium tumefaciens C58, but I confirmed whether it’s true or not. CrdS gene has restriction enzyme sites,EcoRI and PstI.

Biwako-Nagahama E.P tutumi1.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3CrdS12μL
4--
5--
6--
7--
8--
Biwako-Nagahama E.P tutumi2.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3CrdS_nonRE12μL
4CrdS/EcoRⅠ12μL
5CrdS/PstⅠ12μL
6λ-HindⅢ10μL
7500bp DNA ladder10μL
8--

Inverse PCR

Prime STAR MAX25μL
10pmol/μL Primer F1μL
10pmol/μL Primer R1μL
PCR反応液(2ng)1μL
dH2O22μL
Total22μL

CrdS_Fw: AGTACGATCCGCTATTTTCCCG

CrdS_Rv: CAGACCAAGATTTCGCGAACTC

94℃ 2min

↓

98℃ 10sec

48℃ 30sec    35cycles

72℃ 2min

↓

72℃ 2min

TA cloning of CrdS

PCR product1μL
pMD202μL
Ligation kit ver.23μL

16℃ 30min incubate

Ligation of CrdS/pMD20 and Transformation in JM109

Competent cell100μL
DNA6μL

↓Cells were stored on ice for 30min.

↓After 42℃ 30sec heat shock, cells were stored on ice for 2min.

↓Then cells were pre-cultured at 37℃ for 1hr, plated to Ampicillin plate.

Liquid culluture

CrdS/pMD20 21 samples at 37℃,for overnight

MiniPrep of CrdS/pMD20

Linear CrdS/pMD20 DNA :4995bp. This CrdS/pMD20 sample is cccDNA. So, probably picked up CrdS/pMD20.

Biwako-Nagahama E.P tutumi3.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3CrdS No.512μL
4CrdS No.612μL
5CrdS No.812μL
6CrdS No.1512μL
7CrdS No.1712μL
8--

Restriction Enzyme of CrdS/pMD20

CrdS gene had produced clone from Agrobacterium tumefaciens C58, I confirmed whether it’s true or not. CrdS/pMD20 gene has 2 restriction enzyme sites,EcoRI.

Biwako-Nagahama E.P tutumi4.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3CrdS No.512μL
4CrdS No.612μL
5CrdS No.212μL
6CrdS No.1512μL
7CrdS No.1712μL
8--

I comfirmed the direction of CrdS gene.

Sequence of CrdS/pMD20

Biwako-Nagahama Sequences tutumi5.png

PointMutation of CrdS

CrdS gene had Restriction Enzyme Site,EcoRI. We removed the EcoRI site.

Biwako-Nagahama E.P tutumi6.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3CrdS Negative Front Fragment12μL
4CrdS Negative Rear Fragment12μL
5CrdS No.6 Front Fragment12μL
6CrdS No.6 Middle Fragment12μL
7CrdS No.6 Rear Fragment12μL
8CrdS No.15 Front Fragment12μL
9CrdS No.15 Middle Fragment12μL
10CrdS No.15 Rear Fragment12μL
11--
12--
13--
14--
15λ-HindⅢ10μL
16500bp DNA ladder10μL

Front

Fw0 Primer:GGCCGCTTCTAGATGTATTTCAGTGC

Rv1 Primer:CGTTTCGAGGGAGAACTCCAGCG

Middle

Fw1Primer:CGCTGGAGTTCTCCCTCGAAACG

Rv2Primer:CAGCTCATCGGCCTGAAGCGCC

Rear

Fw2 Primer:GGCGCTTCAGGCCGATGAGCTG

Rv0 Primer:GGCGCTACTAGTATTATTATCACCCGAATG

5xPS Buffer10μL
dNTP Mixture4μL
10pmol/µl Primer Fw1 or Fw21μL
10pmol/µl Primer Rv2 or Rv11μL
Templete1μL
Prime STAR HS0.5μL
dH2O32.5μL
Total50μL

94℃ 2min

↓

98℃ 10sec

55℃ 5sec      35cycles

72℃ 70sec

↓

4℃ ∞

Gel Purification of CrdS gene’s Front Fragment and Rear Fragment

Front Fragment DNA and Rear Fragment DNA ,Middle Fragment DNA that 223 bp and 1037 bp ,782 bp band performed Gel Purification by illustra GFX PCR Purification Kit.

CrdS gene’s Front Fragment and Rear Fragment Overlap PCR

Biwako-Nagahama E.P tutumi7.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3CrdS N Front+Middle DNA12μL
4CrdS No.6 Front+Middle DNA12μL
5CrdS No.15 Front+Middle DNA12μL
6λ-HindⅢ10μL
7500bp DNA ladder10μL
8--
Biwako-Nagahama E.P tutumi8.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3CrdS N Front+Rear DNA12μL
4CrdS No.6 Front+Rear DNA12μL
5CrdS No.15 Front+Rear DNA12μL
6λ-HindⅢ10μL
7500bp DNA ladder10μL
8--

N, No.6, No.15 each fragment Overlap PCR completed.

Gel Purification of CrdS

No.8 DNA that about 2ooo bp band performed Gel Purification by illustra GFX PCR Purification Kit.

Adapter PCR of CrdS gene (BioBrick Part)

Biwako-Nagahama E.P tutumi9.png

NoNamevolume
12-log DNA ladder5μL
2λ-HindⅢ6μL
3PCR saample18μL
4--
5--
6--
7--
8--

Fw Primer: GTTTCTTCGAATTCGCGGCCGCTTCTAGATG

Rv Primer: GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATC

Brick Part of CrdS Restrict Enzyme,EcoRI and PstI.

Biwako-Nagahama E.P tutumi10.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3CrdS N F+M non-RE12μL
4CrdS N F+M EcoRⅠ-RE12μL
5CrdS No.6 F+M non-RE12μL
6CrdS No.6 F+M EcoRⅠ-RE12μL
7CrdS No.15 F+M non-RE12μL
8CrdS No.15 F+M EcoRⅠ12μL
9CrdS N F+M+R non-RE12μL
10CrdS N F+M+R PstⅠ-RE12μL
11CrdS No.6 F+M+R non-RE12μL
12Crds No.6 F+M+R PstI RE12μL
13CrdS No.15 F+M+R non-RE12μL
14CrdS No.15 F+M+R PstI RE12μL
15λ-HindⅢ10μL
16500bp DNA ladder10μL
17--
18--
N,No.6,No.15 of CrdS was cut on EcoRI site. Point Mutation of CrdS is unsuccessful.
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