Team:Jilin China/NOTEBOOK

From 2014.igem.org

(Difference between revisions)
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<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
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<html xmlns="http://www.w3.org/1999/xhtml">
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<head>
 +
<meta http-equiv="Content-Type" content="text/html; charset=gb2312" />
 +
<title>无标题文档</title>
 +
</head>
 +
 +
<body>
 +
<h2>The experimental procedure and  analysis</h2>
 +
<h3>1、Dissolving  of primers</h3>
 +
<p>Sterile deionized water whose volume is  determined by primer’s molecular weight is used to dilute the solution of the  primer to 10um/L. And it is prepared for the following experiment.</p>
 +
<h3>2、Mixing  of primers</h3>
 +
<p>Oligonucleotides synthesized in powder  form need to be centrifuged for 1 min at 10000rpm to assemble powders to the  bottom of the EP tube.And be careful to open the cap.<br />
 +
  Make sure that the synthesis report is  consistent to the OD number on the primer label,and check the number of the  dispensing tube before the dissolution of oligonucleotides.The volume of water  added to make 10umol/L oligonucleotides’ solution is calculated according to the  synthetic single report of each primer. Add sterile deionized water and keep it  at room temperature for 2min.And reverse the tube to accelerate the progress of  solubilization. Finally, collect substances at the bottom of the tube after  centrifugation.<br />
 +
  Every six primers are combined to one  group.Add 4ulbeginning fiprimer, 1ulintermediate primer and 4ul end primerto a  new EP tube, then add sterile deionized water to 20ul, mix them up as spare.</p>
 +
<p>&nbsp;</p>
 +
<p>&nbsp;</p>
 +
<div align="center">
 +
  <table border="1" cellspacing="0" cellpadding="0" width="612">
 +
    <tr>
 +
      <td width="67"><p align="center"><strong>组别</strong><strong> </strong></p></td>
 +
      <td width="103"><p align="center"><strong>引物编号</strong><strong> </strong></p></td>
 +
      <td width="65"><p align="center"><strong>吸取量</strong><strong> </strong></p></td>
 +
      <td width="85"><p align="center"><strong>灭菌去离子水添加量</strong><strong> </strong></p></td>
 +
      <td width="75"><p align="center"><strong>总体积</strong><strong> </strong></p></td>
 +
      <td width="108" valign="top"><p align="center"><strong>每</strong><strong>PCR</strong><strong>（</strong><strong>50ul</strong><strong>体系）吸取量</strong><strong> </strong></p></td>
 +
      <td width="108"><p align="center"><strong>终浓度</strong><strong> </strong></p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="67" rowspan="6"><p align="center">A1</p></td>
 +
      <td width="103" valign="top"><p align="center">AC101</p></td>
 +
      <td width="65"><p align="center">4</p></td>
 +
      <td width="85" rowspan="6"><p align="center">8</p></td>
 +
      <td width="75" rowspan="6"><p align="center">20</p></td>
 +
      <td width="108" rowspan="6"><p align="center">5ul</p></td>
 +
      <td width="108"><p align="center">200nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC102</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108"><p align="center">50nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC103</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC104</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC105</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC106</p></td>
 +
      <td width="65"><p align="center">4</p></td>
 +
      <td width="108"><p align="center">200nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="67" rowspan="6"><p align="center">A2</p></td>
 +
      <td width="103" valign="top"><p align="center">AC107</p></td>
 +
      <td width="65"><p align="center">4</p></td>
 +
      <td width="85" rowspan="6"><p align="center">8</p></td>
 +
      <td width="75" rowspan="6"><p align="center">20</p></td>
 +
      <td width="108" rowspan="6"><p align="center">5ul </p></td>
 +
      <td width="108"><p align="center">200nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC108</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108"><p align="center">50nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC109</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC110</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC111</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC112</p></td>
 +
      <td width="65"><p align="center">4</p></td>
 +
      <td width="108"><p align="center">200nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="67" rowspan="6"><p align="center">A3</p></td>
 +
      <td width="103" valign="top"><p align="center">AC113</p></td>
 +
      <td width="65"><p align="center">4</p></td>
 +
      <td width="85" rowspan="6"><p align="center">8</p></td>
 +
      <td width="75" rowspan="6"><p align="center">20</p></td>
 +
      <td width="108" rowspan="6"><p align="center">5ul </p></td>
 +
      <td width="108"><p align="center">200nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC114</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108"><p align="center">50nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC115</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC116</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC117</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC118</p></td>
 +
      <td width="65"><p align="center">4</p></td>
 +
      <td width="108"><p align="center">200nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="67" rowspan="6"><p align="center">A4</p></td>
 +
      <td width="103" valign="top"><p align="center">AC119</p></td>
 +
      <td width="65"><p align="center">4</p></td>
 +
      <td width="85" rowspan="6"><p align="center">8</p></td>
 +
      <td width="75" rowspan="6"><p align="center">20</p></td>
 +
      <td width="108" rowspan="6"><p align="center">5ul </p></td>
 +
      <td width="108"><p align="center">200nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC120</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108"><p align="center">50nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC121</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC122</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC123</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC124</p></td>
 +
      <td width="65"><p align="center">4</p></td>
 +
      <td width="108"><p align="center">200nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="67" rowspan="6"><p align="center">A5</p></td>
 +
      <td width="103" valign="top"><p align="center">AC201</p></td>
 +
      <td width="65"><p align="center">4</p></td>
 +
      <td width="85" rowspan="6"><p align="center">8</p></td>
 +
      <td width="75" rowspan="6"><p align="center">20</p></td>
 +
      <td width="108" rowspan="6"><p align="center">5ul </p></td>
 +
      <td width="108"><p align="center">200nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC202</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108"><p align="center">50nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC203</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC204</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC205</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC206</p></td>
 +
      <td width="65"><p align="center">4</p></td>
 +
      <td width="108"><p align="center">200nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="67" rowspan="6"><p align="center">A6</p></td>
 +
      <td width="103" valign="top"><p align="center">AC207</p></td>
 +
      <td width="65"><p align="center">4</p></td>
 +
      <td width="85" rowspan="6"><p align="center">8</p></td>
 +
      <td width="75" rowspan="6"><p align="center">20</p></td>
 +
      <td width="108" rowspan="6"><p align="center">5ul </p></td>
 +
      <td width="108"><p align="center">200nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC208</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108"><p align="center">50nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC209</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC210</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC211</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC212</p></td>
 +
      <td width="65"><p align="center">4</p></td>
 +
      <td width="108"><p align="center">200nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="67" rowspan="6"><p align="center">A7</p></td>
 +
      <td width="103" valign="top"><p align="center">AC213</p></td>
 +
      <td width="65"><p align="center">4</p></td>
 +
      <td width="85" rowspan="6"><p align="center">8</p></td>
 +
      <td width="75" rowspan="6"><p align="center">20</p></td>
 +
      <td width="108" rowspan="6"><p align="center">5ul </p></td>
 +
      <td width="108"><p align="center">200nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC214</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108"><p align="center">50nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC215</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC216</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC217</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC218</p></td>
 +
      <td width="65"><p align="center">4</p></td>
 +
      <td width="108"><p align="center">200nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="67" rowspan="6"><p align="center">A8</p></td>
 +
      <td width="103" valign="top"><p align="center">AC219</p></td>
 +
      <td width="65"><p align="center">4</p></td>
 +
      <td width="85" rowspan="6"><p align="center">8</p></td>
 +
      <td width="75" rowspan="6"><p align="center">20</p></td>
 +
      <td width="108" rowspan="6"><p align="center">5ul </p></td>
 +
      <td width="108"><p align="center">200nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC220</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108"><p align="center">50nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC221</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC222</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC223</p></td>
 +
      <td width="65"><p align="center">1</p></td>
 +
      <td width="108" valign="top"><p align="center">50nM</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="103" valign="top"><p align="center">AC224</p></td>
 +
      <td width="65"><p align="center">4</p></td>
 +
      <td width="108"><p align="center">200nM</p></td>
 +
    </tr>
 +
  </table>
 +
</div>
 +
 +
 +
 +
<h3>DA-PCR</h3>
 +
<p>Every  six primers which are single-stranded oligonucleotidesare chemically  synthesized were combined to one group. Then they were going to be made as  longer double-stranded fragments of intermediate (Block) by DA-PCR.<br />
 +
  Procedure of DA-PCR： <br />
 +
  Mixed primer solutions                   5μl<br />
 +
  Pfu DNA Polymerase  2.5U              0.5μl     <br />
 +
  dNTP                                4μl<br />
 +
  10×Pfu buffer（Mg2+）               5μl<br />
 +
  ddH20add to 50μl<br />
 +
  Procedure of DA-PCR： <br />
 +
  94℃         2min<br />
 +
  94℃         30S<br />
 +
  50℃         30S     25 cycles<br />
 +
  72℃         1min<br />
 +
  72℃         10min<br />
 +
  4℃preservation<br />
 +
  End<br />
 +
  DA-PCR splicing reaction product was  detected in 2% agarose gel electrophoresis,<br />
 +
  DA-PCR product              5μl <br />
 +
  10×Loading Buffer         0.6μl    spotting after mixed<br />
 +
  100bp Marker              5μl    spotting directly<br />
 +
  75V electrophoress for 1h。 </p>
 +
<h3>4、OE-PCR </h3>
 +
<p>    Take the 5 double strand intermediate  fragment (Block) spliced by DA-PCR as templates, blocks were further connected  by OR-PCR, OE-PCR in a reaction system consisting of:<br />
 +
  Block1                       1μl<br />
 +
  Block2                       1μl<br />
 +
  Block3                       1μl<br />
 +
  Block4                       1μl<br />
 +
  Block5                       1μl<br />
 +
  Block6                       1μl<br />
 +
  Block7                       1μl<br />
 +
  Block8                       1μl <br />
 +
  AC1                         1μl<br />
 +
  AC4                         1μl<br />
 +
  <em>Pfu</em> DNA Polymerase 2.5U       0.5μl <br />
 +
  dNTP                        4μl<br />
 +
  10×<em>Pfu</em> buffer（Mg2+）         5μl<br />
 +
  <img width="350" height="2" src="file:///C|/Users/Administrator/AppData/Roaming/Macromedia/Dreamweaver 8/OfficeImageTemp/clip_image001_0000.gif" />Sterilized  ultrapure water   to 50ul<br />
 +
  Procedure of OE-PCR： <br />
 +
  94℃         2min<br />
 +
  <img width="14" height="65" src="file:///C|/Users/Administrator/AppData/Roaming/Macromedia/Dreamweaver 8/OfficeImageTemp/clip_image002.gif" />        94℃         30S<br />
 +
  50℃         30S    20 cycles<br />
 +
  72℃        2min<br />
 +
  72℃       10min<br />
 +
  <img width="350" height="2" src="file:///C|/Users/Administrator/AppData/Roaming/Macromedia/Dreamweaver 8/OfficeImageTemp/clip_image003.gif" />        4℃preservation<br />
 +
  End<br />
 +
  The full-length gene  spliced by OE-PCR was detected by a 1% agarose gel electrophoresis, specific  programs are as follows<br />
 +
  <img width="14" height="35" src="file:///C|/Users/Administrator/AppData/Roaming/Macromedia/Dreamweaver 8/OfficeImageTemp/clip_image004.gif" />OE-PCR  product           5μl <br />
 +
  10×Loading Buffer        0.6μl    spotted after mixing well<br />
 +
  <img width="350" height="2" src="file:///C|/Users/Administrator/AppData/Roaming/Macromedia/Dreamweaver 8/OfficeImageTemp/clip_image003_0000.gif" />100bp  Marker              5μl   spotted directly<br />
 +
  80V electrophoresis for  1h。 </p>
 +
<h3>5、Constructing  and sequencing of subcloning vector </h3>
 +
<p>We design a primer AC-B with Blp I site  and a primer AC-S with SgrA I site. Then we use RFP-promoter-GFP as template in  PCR. Then we use Blp I and SgrA I enzyme to digest the target fragment and  pET32a vector(37℃,3h) and link it overnight(16℃). After this step, transform the  linked vector to competent cell(E.coli BL21) and select the desirable colony by  using “Blue-White Selection” method.<br />
 +
    <strong>Constitute of </strong><em>Blp</em>Ⅰand <em>Sgr</em>AⅠdouble  enzyme digestion reaction system:<strong> </strong><br />
 +
  Gene or plasmid                   10μl<br />
 +
  <em>Blp</em>Ⅰ              0.3μl<br />
 +
  <em>Sgr</em>AⅠ0.3μl<br />
 +
  10×NEBuffer 2.12μl<br />
 +
  Sterilizing  ultrapure watertop up to20μl<br />
 +
  <strong>Constitute of linking reaction system</strong><strong>：</strong><strong> </strong><br />
 +
  RFP-promoter-GFP                    10μl<br />
 +
  pET32a                              3μl<br />
 +
  T4  ligase                            0.5μl<br />
 +
  10×T4  ligase Buffer                   2μl<br />
 +
  Sterilizing  ultrapure water           top up to 20μl <br />
 +
  Preparation process of competent cell <strong><em>E.coli</em>BL21</strong><strong>：</strong><strong> </strong><br />
 +
  (1) Culturing strain <em>E.coli</em>BL21(37℃)overnight  to recovery culture, Adding overnight bacteria culture fluid 600uLto 30 mL LB  culture medium, and 37℃，200  r/min shake culturing 1-2 h, until OD600 is around 0.3-0.4.<br />
 +
  (2) In the aseptic  condition, transfer 30mLbacteria fluid to the centrifugal tube in the ice 10  min to cooling the culture to 0℃.<br />
 +
  (3)  Centrifuging 10 min in precooling centrifugal machine over 4000r/min, throw  supernate, and then add30mLprecooling 0.1mol/L CaCl2 solution to  resuspension precipitation in the ice 10 min.<br />
 +
  (4) Centrifuging 4000r/min  in 4℃10min, throw  supernate,  each 30mL initial culture  resuspension cell precipitation with 1.0mL precooling 0.1mol/LCaCl2  solution, and then 200μL each one.<br />
 +
  <strong>Heal  shock transformation process</strong><strong>：</strong><strong> </strong><br />
 +
  (1) Thawing freshly  prepared or -80℃ competentcellBL21suspension.<br />
 +
  (2) Add constructed  recombinant cloning vector plasmidspSB1C3-A(no more than 50ng, 10μl), shake  gently, and then in the ice 30min.<br />
 +
  (3)  Put EP tube in 42℃ water bath90s，and  then transfer it to ice water bath to cool cells1-2 min.<br />
 +
  (4) add 800μL LB medium  to EP tube，transfer  it to 37℃  table，150r/min，to  recovery strain 45 min.<br />
 +
  Spread plate and  Culture<br />
 +
  (1)After culture in 37℃,  centrifuge the cell solution(5000rpm,5min) and concentrate it to 200μl.  Then coat it to LB culture with chloramphenicol and set a positive control.<br />
 +
  (2) Wait until the  solution in culture is full absorbed by cell, then invert the plate and culture  it in 37℃  overnight.<br />
 +
  Sequence test<br />
 +
  On the pET32a vector(as shown in Fig 1), use the  RFP-promoter-GFP gene (1535bp) to replace the fragment between <em>Blp</em>Ⅰ site and <em>Sgr</em>A I site (753bp) and then we get  the reconstructed plasmid, pET-mlr promoter.</p>
 +
 +
 +
</table>
</table>
</div>
</div>
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
<!----
<!----
<h2>二、实验操作及结果分析 </h2>
<h2>二、实验操作及结果分析 </h2>

Revision as of 02:12, 18 October 2014

Welcome!
Team Jilin China
NOTEBOOK

Synthesis and Characterisation of the Mlr Promoter

Research background

A novel pathway for degradation of the cyanobacterial heptapeptide hepatotoxin microcystin LR was identified in a newly isolated Sphingomonas sp. (Bourne et al. 1996 Appl. Environ. Microbiol. 62: 4086–4094). And the gene cluster involved in bacterial degradation of the cyanobacterial
toxin microcystin LR were be reported by David G. Bourne in 2001. The cloning and molecular characterisation of four genes from this Sphingomonas sp. that exist on a 5.8-kb genomic fragment and encode the three hydrolytic enzymes involved in this pathway together with a putative oligopeptide transporter. Situated immediately downstream of mlrA with the same direction of transcription is a gene mlrD, whose conceptual translation (MlrD, 442residues) shows significant sequence identity and similar potential transmembrane spanning regions to the PTR2family of oligopeptide transporters. A gene mlrB is situated downstream of the mlrA and mlrD genes, but transcribed in the opposite direction. The gene encodes the enzyme MlrB (402residues) which cleaves linear microcystin LR to a tetrapeptide degradation product. This enzyme belongs to the “penicillin-binding enzyme” family of active site serine hydrolases. The final gene in the cluster mlrC, is located upstream of the mlrA gene and is transcribed in the opposite direction. It codes for MlrC (507 residues) which mediates further peptidolytic degradation of the tetrapeptide. This protein shows significant sequence identity to a hypothetical protein from Streptomyces coelicolor. It is suspected to be a metallopeptidase based on inhibition by metal chelators. It is postulated on the basis of comparison with other microorganisms that the genes in this cluster may all be involved in cell wall peptidoglycan cycling and subsequently act fortuitously in hydrolysis of microcystin LR.
In this study, we  synthesized a RFP-promoter-GFP sequence to detect the relationship between MLR promoter and microcystin LR. This synthesized sequence using the promoter sequence between mlrA and mlrC, adding the red fluorescent protein sequence in the upstream to instead mlrC gene, and adding green fluorescent protein sequence in the downstream to instead mlrA gene.

Fig 1 Restriction map of the 5.8 kb of sequence data and localisation of genes involved in microcystin LR degradation.

methods

Synthetic sequence

    BamHI
GCCTGCAGctataaaaataaatgatgtctaccttctgttctttcatattgttcaacaattgtataatcttcattatgtgatgtaatatctaacttagcatctacataataatatcctggtaattgaactggttttttagccatataaattgatttaaattcaactaaataatgtcctccgtcctttaattttagagctttatgtgtttctccctttaaaaccccgtctcttggatataatctttctgtactagcttcccatcccattgtttttttttgcataactggtccgtctgatggaaaattaaccccaataaatttaactttataaataaaacaaccatcttgtaatgatgaatcttgtgtaactgtagcaactccaccatcttcaaaattcataactctttcccatttaaatccttctggaaaagataattttttataatctggaatatcagctggatgtttaacgtaaacctttgatccatattgaaattgtggtgataaaatatcccacgcaaatggtaatggtcctccttttgtaacctttaatttaactgtattatgtccttcataCggtctaccttctccttctccctcaatttcaaattcatgtccattaactgtaccttccattctaactttaaatctcataaattctgtaataacattttctgatgaagccatAacacgagtcttcggtttgctgttgtttgcaccagctgctgcatcttcaccccataaatcaaccgaacggacagtcaattcttgttgaccatcctgggcctatctgaaatgttctgctgacagaacgggagaattgaaccatgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcaCatgaaacagcatgactttttcaagagtgccatgcctgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataaGAATTCCG
EcoRI

Sequence analysis

 _Base Count : 1551 bp (489 A, 496 T, 314 C, 252 G)
 _Composition : 36% GC, 64% AT

Absent Sites
AarI AatII Acc65I AciI AclI AfeI AflII AgeI AhdI AleI ApaI ApaLI AscI AseI AsiSI AvaI BamHI BanI BanII BbvCI BceAI BciVI BclI BfuAI BglI BglII BlpI BmgBI BmrI BmtI Bpu10I BsaBI BsaHI BsaWI BseMII BseRI BsgI BsiEI BsiHKAI BsiWI BsmI BsoBI BspCNI BspEI BspHI BspMI BspQI BsrBI BsrDI BsrFI BssHII BstEII BstUI Bsu36I BtrI BtsI Cac8I ClaI DraIII EagI EarI EciI Eco53KI EcoNI EcoO109I EcoRV FauI FseI FspAI FspI HaeII HgaI HhaI HinP1I HindIII HpaII Hpy99I KasI KpnI MluI MmeI NaeI NarI NciI NdeI NgoMIV NheI NlaIV NmeAIII NotI NruI NsiI NspI PacI PasI PciI PflFI PflMI PfoI PmeI PpuMI PshAI PspOMI PspXI PstI PvuI RsrII SacI SacII SalI SanDI SbfI ScaI SexAI SfiI SfoI SgrAI SmaI SnaBI SpeI SphI SrfI SspI StuI TauI TspMI Tth111I XbaI XcmI XhoI XmaI XmnI ZraI
Unique Sites
Unique Sites
BamHI(3)
DdeI (92)
Esp3I (223)
SfcI (367)
BstXI (512) MslI (513)
BssSI (688) PleI (691) BbsI (693)
MwoI (714)
BpmI (851)
BsmFI (859)
Bsp1286I (895)
BtgI (991) MscI (994) Tsp45I (1006)
BsrGI (1101)
AflIII (1147) BsaAI (1148)
BstZ17I (1273)
HpaI (1312)
BtgZI (1391)
AsuII (1446) BstYI (1452) BsaI (1469) DrdI (1471)
SmlI (1486) BseYI (1504)
EcoRI (1543)

Building Block Design

The maximum allowable assembly oligo length is equal to the target assembly oligo length (60). This may cause some weird behavior, especially in terms of overlap melting temperature.

2 building blocks were generated.
Building Block mlrAC.1   789bp   1..789 
Left  - 5' GCCCTAGGCTATAAAAATAAATGATG 3'
Rght  - 5' ATAGGCCCAGGATGGTCAA 3'
RghtU - 5' ATAGGCCCAGGAUGGTCAA 3'
Sequence:
Assembly Oligos: average overlap Tm is 48°;average oligo length is 56bp.

GCCCTAGGCTATAAAAATAAATGATGTCTACCTTCTGTTCTTTCATATTGTTCAACAATTGTATAATCTTCATTATGTGATGTAATATCTAACTTAGCATCTACATAATAATATCCTGGTAATTGAACTGGTTTTTTAGCCATATAAATTGATTTAAATTCAACTAAATAATGTCCTCCGTCCTTTAATTTTAGAGCTTTATGTGTTTCTCCCTTTAAAACCCCGTCTCTTGGATATAATCTTTCTGTACTAGCTTCCCATCCCATTGTTTTTTTTTGCATAACTGGTCCGTCTGATGGAAAATTAACCCCAATAAATTTAACTTTATAAATAAAACAACCATCTTGTAATGATGAATCTTGTGTAACTGTAGCAACTCCACCATCTTCAAAATTCATAACTCTTTCCCATTTAAATCCTTCTGGAAAAGATAATTTTTTATAATCTGGAATATCAGCTGGATGTTTAACGTAAACCTTTGATCCATATTGAAATTGTGGTGATAAAATATCCCACGCAAATGGTAATGGTCCTCCTTTTGTAACCTTTAATTTAACTGTATTATGTCCTTCATACGGTCTACCTTCTCCTTCTCCCTCAATTTCAAATTCATGTCCATTAACTGTACCTTCCATTCTAACTTTAAATCTCATAAATTCTGTAATAACATTTTCTGATGAAGCCATAACACGAGTCTTCGGTTTGCTGTTGTTTGCACCAGCTGCTGCATCTTCACCCCATAAATCAACCGAACGGACAGTCAATTCTTGTTGACCATCCTGGGCCTAT
GCCCTAGGCTATAAAAATAAATGATGTCTACCTTCTGTTCTTTCATATTGTTCAACAAT      ATCTTCATTATGTGATGTAATATCTAACTTAGCATCTACATAATAATATCCTGGTAAT      GGTTTTTTAGCCATATAAATTGATTTAAATTCAACTAAATAATGTCCTCCGTCC          GAGCTTTATGTGTTTCTCCCTTTAAAACCCCGTCTCTTGGATATAATCTTTCTGTAC       CCATCCCATTGTTTTTTTTTGCATAACTGGTCCGTCTGATGGAAAATTAACCC           ACTTTATAAATAAAACAACCATCTTGTAATGATGAATCTTGTGTAACTGTAGCA          CTTCAAAATTCATAACTCTTTCCCATTTAAATCCTTCTGGAAAAGATAATTTTTTATAAT    AATATCAGCTGGATGTTTAACGTAAACCTTTGATCCATATTGAAATTGTGGTGATAAA      CACGCAAATGGTAATGGTCCTCCTTTTGTAACCTTTAATTTAACTGTATTATGTCCTTC     GTCTACCTTCTCCTTCTCCCTCAATTTCAAATTCATGTCCATTAACTGTACCTT          TTTAAATCTCATAAATTCTGTAATAACATTTTCTGATGAAGCCATAACACGA            CTGTTGTTTGCACCAGCTGCTGCATCTTCACCCCATAAATCAACCGAACGGA                                
                                 AGACAAGAAAGTATAACAAGTTGTTAACATATTAGAAGTAATACACTACATTATAGATTG    GTAGATGTATTATTATAGGACCATTAACTTGACCAAAAAATCGGTATATTTAACTAAATT    TTGATTTATTACAGGAGGCAGGAAATTAAAATCTCGAAATACACAAAGAGGG            AGAGAACCTATATTAGAAAGACATGATCGAAGGGTAGGGTAACAAAAAAAAACG          GCAGACTACCTTTTAATTGGGGTTATTTAAATTGAAATATTTATTTTGTTGGTAGAACAT    ACTTAGAACACATTGACATCGTTGAGGTGGTAGAAGTTTTAAGTATTGAGAAAGGG        GGAAGACCTTTTCTATTAAAAAATATTAGACCTTATAGTCGACCTACAAATTGC          TAGGTATAACTTTAACACCACTATTTTATAGGGTGCGTTTACCATTACCAGG            GAAATTAAATTGACATAATACAGGAAGTATGCCAGATGGAAGAGGAAGAGGG            AGTACAGGTAATTGACATGGAAGGTAAGATTGAAATTTAGAGTATTTAAGACATTATTGT    GACTACTTCGGTATTGTGCTCAGAAGCCAAACGACAACAAACGTGGTCGACG            GGTATTTAGTTGGCTTGCCTGTCAGTTAAGAACAACTGGTAGGACCCGGATA
CGGGATCCGATATTTTTATTTACTACAGATGGAAGACAAGAAAGTATAACAAGTTGTTAACATATTAGAAGTAATACACTACATTATAGATTGAATCGTAGAT

CGGGATCCGATATTTTTATTTACTACAGATGGAAGACAAGAAAGTATAACAAGTTGTTAACATATTAGAAGTAATACACTACATTATAGATTGAATCGTAGATGTATTATTATAGGACCATTAACTTGACCAAAAAATCGGTATATTTAACTAAATTTAAGTTGATTTATTACAGGAGGCAGGAAATTAAAATCTCGAAATACACAAAGAGGGAAATTTTGGGGCAGAGAACCTATATTAGAAAGACATGATCGAAGGGTAGGGTAACAAAAAAAAACGTATTGACCAGGCAGACTACCTTTTAATTGGGGTTATTTAAATTGAAATATTTATTTTGTTGGTAGAACATTACTACTTAGAACACATTGACATCGTTGAGGTGGTAGAAGTTTTAAGTATTGAGAAAGGGTAAATTTAGGAAGACCTTTTCTATTAAAAAATATTAGACCTTATAGTCGACCTACAAATTGCATTTGGAAACTAGGTATAACTTTAACACCACTATTTTATAGGGTGCGTTTACCATTACCAGGAGGAAAACATTGGAAATTAAATTGACATAATACAGGAAGTATGCCAGATGGAAGAGGAAGAGGGAGTTAAAGTTTAAGTACAGGTAATTGACATGGAAGGTAAGATTGAAATTTAGAGTATTTAAGACATTATTGTAAAAGACTACTTCGGTATTGTGCTCAGAAGCCAAACGACAACAAACGTGGTCGACGACGTAGAAGTGGGGTATTTAGTTGGCTTGCCTGTCAGTTAAGAACAACTGGTAGGACCCGGATA    .
Building Block mlrAC.2   775bp   777..1551 
Left  - 5' ATCCTGGGCCTATCTGAAATG 3'
LeftU - 5' ATCCTGGGCCTAUCTGAAATG 3'
Rght  - 5' CGGAATTCTTATTTGTATAGTTCATCC 3'
Sequence:
Assembly Oligos: average overlap Tm is 49°;average oligo length is 53bp.
ATCCTGGGCCTATCTGAAATGTTCTGCTGACAGAACGGGAGAATTGAACCATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCACATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCTGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAAGAATTCCG
ATCCTGGGCCTATCTGAAATGTTCTGCTGACAGAACGGGAGAATTGAACCAT            AGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGG       TTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTT          GCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGG            ATGCTTTGCGAGATACCCAGATCACATGAAACAGCATGACTTTTTCAAGAGTGC          GGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGA           GTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTT     AGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATA     TGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTG     GGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGG         TTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCC           GAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATG                               
                                CTTGCCCTCTTAACTTGGTACTCATTTCCTCTTCTTGAAAAGTGACCTCAACAG          AACTTAATCTACCACTACAATTACCCGTGTTTAAAAGACAGTCACCTCTCCC            TTGTATGCCTTTTGAATGGGAATTTAAATAAACGTGATGACCTTTTGATGGA            TGTGAACAGTGATGAAAGCCAATACCACAAGTTACGAAACGCTCTATGGGTC            TCGTACTGAAAAAGTTCTCACGGTACGGACTTCCAATACATGTCCTTTCTTGAT          TCTACTGCCCTTGATGTTCTGTGCACGACTTCAGTTCAAACTTCCACTATGG          CTTAGCTCAATTTTCCATAACTAAAATTTCTTCTACCTTTGTAAGAACCTGTG         ATGTTGATATTGAGTGTGTTACATATGTAGTACCGTCTGTTTGTTTTCTTACC         ATTGAAGTTTTAATCTGTGTTGTAACTTCTACCTTCGCAAGTTGATCGTCT           TTTTATGAGGTTAACCGCTACCGGGACAGGAAAATGGTCTGTTGGTAATGGAC         AGACGGGAAAGCTTTCTAGGGTTGCTTTTCTCTCTGGTGTACCAGGAAGAA           TCGACGACCCTAATGTGTACCGTACCTACTTGATATGTTTATTCTTAAGGC
TAGGACCCGGATAGACTTTACAAGACGACTGTCTTGCCCTCTTAACTTGGTACTCATTTCCTCTTCTTGAAAAGTGACCTCAACAGGGTTAAGAACAACTTAATCTACCACTACAATTACCCGTGTTTAAAAGACAGTCACCTCTCCCACTTCCACTACGTTGTATGCCTTTTGAATGGGAATTTAAATAAACGTGATGACCTTTTGATGGACAAGGTACCGGTTGTGAACAGTGATGAAAGCCAATACCACAAGTTACGAAACGCTCTATGGGTCTAGTGTACTTTGTCGTACTGAAAAAGTTCTCACGGTACGGACTTCCAATACATGTCCTTTCTTGATATAAAAAGTTTCTACTGCCCTTGATGTTCTGTGCACGACTTCAGTTCAAACTTCCACTATGGGAACAATTATCTTAGCTCAATTTTCCATAACTAAAATTTCTTCTACCTTTGTAAGAACCTGTGTTTAACCTTATGTTGATATTGAGTGTGTTACATATGTAGTACCGTCTGTTTGTTTTCTTACCTTAGTTTCAATTGAAGTTTTAATCTGTGTTGTAACTTCTACCTTCGCAAGTTGATCGTCTGGTAATAGTTGTTTTATGAGGTTAACCGCTACCGGGACAGGAAAATGGTCTGTTGGTAATGGACAGGTGTGTTAGACGGGAAAGCTTTCTAGGGTTGCTTTTCTCTCTGGTGTACCAGGAAGAACTCAAACATTGTCGACGACCCTAATGTGTACCGTACCTACTTGATATGTTTATTCTTAAGGC 

Primer synthesis

Primer synthesis order o Comate Bioscience Co.,Ltd.

Num

Sequence(5' to 3')

Base Count（bp）

Total Synthesis(OD)

Subpackage Pipe Counts

Purification Mode

AC101

GCCCTAGGCTATAAAAATAAATGATGTCTACCTTCTGTTCTTTCATATTGTTCAACAAT

59

1

1

PAGE

AC102

GTTAGATATTACATCACATAATGAAGATTATACAATTGTTGAACAATATGAAAGAACAGA

60

1

1

PAGE

AC103

ATCTTCATTATGTGATGTAATATCTAACTTAGCATCTACATAATAATATCCTGGTAAT

58

1

1

PAGE

AC104

TTAAATCAATTTATATGGCTAAAAAACCAGTTCAATTACCAGGATATTATTATGTAGATG

60

1

1

PAGE

AC105

GGTTTTTTAGCCATATAAATTGATTTAAATTCAACTAAATAATGTCCTCCGTCC

54

1

1

PAGE

AC106

GGGAGAAACACATAAAGCTCTAAAATTAAAGGACGGAGGACATTATTTAGTT

52

1

1

PAGE

AC107

GAGCTTTATGTGTTTCTCCCTTTAAAACCCCGTCTCTTGGATATAATCTTTCTGTAC

57

1

1

PAGE

AC108

GCAAAAAAAAACAATGGGATGGGAAGCTAGTACAGAAAGATTATATCCAAGAGA

54

1

1

PAGE

AC109

CCATCCCATTGTTTTTTTTTGCATAACTGGTCCGTCTGATGGAAAATTAACCC

53

1

1

PAGE

AC110

TACAAGATGGTTGTTTTATTTATAAAGTTAAATTTATTGGGGTTAATTTTCCATCAGACG

60

1

1

PAGE

AC111

ACTTTATAAATAAAACAACCATCTTGTAATGATGAATCTTGTGTAACTGTAGCA

54

1

1

PAGE

AC112

GGGAAAGAGTTATGAATTTTGAAGATGGTGGAGTTGCTACAGTTACACAAGATTCA

56

1

1

PAGE

AC113

CTTCAAAATTCATAACTCTTTCCCATTTAAATCCTTCTGGAAAAGATAATTTTTTATAAT

60

1

1

PAGE

AC114

CGTTAAACATCCAGCTGATATTCCAGATTATAAAAAATTATCTTTTCCAGAAGG

54

1

1

PAGE

AC115

AATATCAGCTGGATGTTTAACGTAAACCTTTGATCCATATTGAAATTGTGGTGATAAA

58

1

1

PAGE

AC116

GGACCATTACCATTTGCGTGGGATATTTTATCACCACAATTTCAATATGGAT

52

1

1

PAGE

AC117

CACGCAAATGGTAATGGTCCTCCTTTTGTAACCTTTAATTTAACTGTATTATGTCCTTC

59

1

1

PAGE

AC118

GGGAGAAGGAGAAGGTAGACCGTATGAAGGACATAATACAGTTAAATTAAAG

52

1

1

PAGE

AC119

GTCTACCTTCTCCTTCTCCCTCAATTTCAAATTCATGTCCATTAACTGTACCTT

54

1

1

PAGE

AC120

TGTTATTACAGAATTTATGAGATTTAAAGTTAGAATGGAAGGTACAGTTAATGGACATGA

60

1

1

PAGE

AC121

TTTAAATCTCATAAATTCTGTAATAACATTTTCTGATGAAGCCATAACACGA

52

1

1

PAGE

AC122

GCAGCTGGTGCAAACAACAGCAAACCGAAGACTCGTGTTATGGCTTCATCAG

52

1

1

PAGE

AC123

CTGTTGTTTGCACCAGCTGCTGCATCTTCACCCCATAAATCAACCGAACGGA

52

1

1

PAGE

AC124

ATAGGCCCAGGATGGTCAACAAGAATTGACTGTCCGTTCGGTTGATTTATGG

52

1

1

PAGE

AC201

ATCCTGGGCCTATCTGAAATGTTCTGCTGACAGAACGGGAGAATTGAACCAT

52

1

1

PAGE

AC202

GACAACTCCAGTGAAAAGTTCTTCTCCTTTACTCATGGTTCAATTCTCCCGTTC

54

1

1

PAGE

AC203

AGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGG

57

1

1

PAGE

AC204

CCCTCTCCACTGACAGAAAATTTGTGCCCATTAACATCACCATCTAATTCAA

52

1

1

PAGE

AC205

TTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTT

54

1

1

PAGE

AC206

AGGTAGTTTTCCAGTAGTGCAAATAAATTTAAGGGTAAGTTTTCCGTATGTT

52

1

1

PAGE

AC207

GCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGG

52

1

1

PAGE

AC208

CTGGGTATCTCGCAAAGCATTGAACACCATAACCGAAAGTAGTGACAAGTGT

52

1

1

PAGE

AC209

ATGCTTTGCGAGATACCCAGATCACATGAAACAGCATGACTTTTTCAAGAGTGC

54

1

1

PAGE

AC210

TAGTTCTTTCCTGTACATAACCTTCAGGCATGGCACTCTTGAAAAAGTCATGCT

54

1

1

PAGE

AC211

GGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGA

52

1

1

PAGE

AC212

GGTATCACCTTCAAACTTGACTTCAGCACGTGTCTTGTAGTTCCCGTCATCT

52

1

1

PAGE

AC213

GTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTT

57

1

1

PAGE

AC214

GTGTCCAAGAATGTTTCCATCTTCTTTAAAATCAATACCTTTTAACTCGATTC

53

1

1

PAGE

AC215

AGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATA

57

1

1

PAGE
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The experimental procedure and analysis

1、Dissolving of primers

Sterile deionized water whose volume is determined by primer’s molecular weight is used to dilute the solution of the primer to 10um/L. And it is prepared for the following experiment.

2、Mixing of primers

Oligonucleotides synthesized in powder form need to be centrifuged for 1 min at 10000rpm to assemble powders to the bottom of the EP tube.And be careful to open the cap.
Make sure that the synthesis report is consistent to the OD number on the primer label,and check the number of the dispensing tube before the dissolution of oligonucleotides.The volume of water added to make 10umol/L oligonucleotides’ solution is calculated according to the synthetic single report of each primer. Add sterile deionized water and keep it at room temperature for 2min.And reverse the tube to accelerate the progress of solubilization. Finally, collect substances at the bottom of the tube after centrifugation.
Every six primers are combined to one group.Add 4ulbeginning fiprimer, 1ulintermediate primer and 4ul end primerto a new EP tube, then add sterile deionized water to 20ul, mix them up as spare.

 

 

AC215

AGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATA

57

1

1

PAGE

AC216

CCATTCTTTTGTTTGTCTGCCATGATGTATACATTGTGTGAGTTATAGTTGTA

53

1

1

PAGE

AC217

TGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTG

57

1

1

PAGE

AC218

TCTGCTAGTTGAACGCTTCCATCTTCAATGTTGTGTCTAATTTTGAAGTTA

51

1

1

PAGE

AC219

GGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGG

53

1

1

PAGE

AC220

CAGGTAATGGTTGTCTGGTAAAAGGACAGGGCCATCGCCAATTGGAGTATTTT

53

1

1

PAGE

AC221

TTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCC

51

1

1

PAGE

AC222

AAGAAGGACCATGTGGTCTCTCTTTTCGTTGGGATCTTTCGAAAGGGCAGA

51

1

1

PAGE

AC223

GAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATG

51

1

1

PAGE

AC224

CGGAATTCTTATTTGTATAGTTCATCCATGCCATGTGTAATCCCAGCAGCT

51

1

1

PAGE

AC1

GCCCTAGGCTATAAAAATAAATGATG

26

1

1

PAGE

AC2

ATAGGCCCAGGATGGTCAA

19

1

1

PAGE

AC3

ATCCTGGGCCTATCTGAAATG

21

1

1

PAGE

AC4

CGGAATTCTTATTTGTATAGTTCATCC

27

1

1

PAGE

组别

引物编号

吸取量

灭菌去离子水添加量

总体积

每PCR（50ul体系）吸取量

终浓度

A1

AC101

4

8

20

5ul

200nM

AC102

1

50nM

AC103

1

50nM

AC104

1

50nM

AC105

1

50nM

AC106

4

200nM

A2

AC107

4

8

20

5ul

200nM

AC108

1

50nM

AC109

1

50nM

AC110

1

50nM

AC111

1

50nM

AC112

4

200nM

A3

AC113

4

8

20

5ul

200nM

AC114

1

50nM

AC115

1

50nM

AC116

1

50nM

AC117

1

50nM

AC118

4

200nM

A4

AC119

4

8

20

5ul

200nM

AC120

1

50nM

AC121

1

50nM

AC122

1

50nM

AC123

1

50nM

AC124

4

200nM

A5

AC201

4

8

20

5ul

200nM

AC202

1

50nM

AC203

1

50nM

AC204

1

50nM

AC205

1

50nM

AC206

4

200nM

A6

AC207

4

8

20

5ul

200nM

AC208

1

50nM

AC209

1

50nM

AC210

1

50nM

AC211

1

50nM

AC212

4

200nM

A7

AC213

4

8

20

5ul

200nM

AC214

1

50nM

AC215

1

50nM

AC216

1

50nM

AC217

1

50nM

AC218

4

200nM

A8

AC219

4

8

20

5ul

200nM

AC220

1

50nM

AC221

1

50nM

AC222

1

50nM

AC223

1

50nM

AC224

4

200nM

DA-PCR

Every six primers which are single-stranded oligonucleotidesare chemically synthesized were combined to one group. Then they were going to be made as longer double-stranded fragments of intermediate (Block) by DA-PCR.
Procedure of DA-PCR：
Mixed primer solutions                  5μl
Pfu DNA Polymerase 2.5U              0.5μl    
dNTP                                4μl
10×Pfu buffer（Mg2+）               5μl
ddH20add to 50μl
Procedure of DA-PCR：
94℃         2min
94℃         30S
50℃         30S    25 cycles
72℃         1min
72℃        10min
4℃preservation
End
DA-PCR splicing reaction product was detected in 2% agarose gel electrophoresis,
DA-PCR product              5μl
10×Loading Buffer         0.6μl   spotting after mixed
100bp Marker              5μl    spotting directly
75V electrophoress for 1h。

4、OE-PCR

    Take the 5 double strand intermediate fragment (Block) spliced by DA-PCR as templates, blocks were further connected by OR-PCR, OE-PCR in a reaction system consisting of:
Block1                       1μl
Block2                       1μl
Block3                       1μl
Block4                       1μl
Block5                       1μl
Block6                       1μl
Block7                       1μl
Block8                       1μl
AC1                         1μl
AC4                        1μl
Pfu DNA Polymerase 2.5U       0.5μl
dNTP                        4μl
10×Pfu buffer（Mg2+）         5μl
Sterilized ultrapure water   to 50ul
Procedure of OE-PCR：
94℃         2min
        94℃         30S
50℃         30S   20 cycles
72℃        2min
72℃       10min
        4℃preservation
End
The full-length gene spliced by OE-PCR was detected by a 1% agarose gel electrophoresis, specific programs are as follows
OE-PCR product           5μl
10×Loading Buffer        0.6μl   spotted after mixing well
100bp Marker              5μl   spotted directly
80V electrophoresis for 1h。

5、Constructing and sequencing of subcloning vector

We design a primer AC-B with Blp I site and a primer AC-S with SgrA I site. Then we use RFP-promoter-GFP as template in PCR. Then we use Blp I and SgrA I enzyme to digest the target fragment and pET32a vector(37℃,3h) and link it overnight(16℃). After this step, transform the linked vector to competent cell(E.coli BL21) and select the desirable colony by using “Blue-White Selection” method.
Constitute of BlpⅠand SgrAⅠdouble enzyme digestion reaction system:
Gene or plasmid                   10μl
BlpⅠ              0.3μl
SgrAⅠ0.3μl
10×NEBuffer 2.12μl
Sterilizing ultrapure watertop up to20μl
Constitute of linking reaction system：
RFP-promoter-GFP                    10μl
pET32a                              3μl
T4 ligase                            0.5μl
10×T4 ligase Buffer                   2μl
Sterilizing ultrapure water           top up to 20μl
Preparation process of competent cell E.coliBL21：
(1) Culturing strain E.coliBL21(37℃)overnight to recovery culture, Adding overnight bacteria culture fluid 600uLto 30 mL LB culture medium, and 37℃，200 r/min shake culturing 1-2 h, until OD600 is around 0.3-0.4.
(2) In the aseptic condition, transfer 30mLbacteria fluid to the centrifugal tube in the ice 10 min to cooling the culture to 0℃.
(3) Centrifuging 10 min in precooling centrifugal machine over 4000r/min, throw supernate, and then add30mLprecooling 0.1mol/L CaCl2 solution to resuspension precipitation in the ice 10 min.
(4) Centrifuging 4000r/min in 4℃10min, throw supernate,  each 30mL initial culture resuspension cell precipitation with 1.0mL precooling 0.1mol/LCaCl2 solution, and then 200μL each one.
Heal shock transformation process：
(1) Thawing freshly prepared or -80℃ competentcellBL21suspension.
(2) Add constructed recombinant cloning vector plasmidspSB1C3-A(no more than 50ng, 10μl), shake gently, and then in the ice 30min.
(3) Put EP tube in 42℃ water bath90s，and then transfer it to ice water bath to cool cells1-2 min.
(4) add 800μL LB medium to EP tube，transfer it to 37℃ table，150r/min，to recovery strain 45 min.
Spread plate and Culture
(1)After culture in 37℃, centrifuge the cell solution(5000rpm,5min) and concentrate it to 200μl. Then coat it to LB culture with chloramphenicol and set a positive control.
(2) Wait until the solution in culture is full absorbed by cell, then invert the plate and culture it in 37℃ overnight.
Sequence test
On the pET32a vector(as shown in Fig 1), use the RFP-promoter-GFP gene (1535bp) to replace the fragment between BlpⅠ site and SgrA I site (753bp) and then we get the reconstructed plasmid, pET-mlr promoter.

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