Team:Nankai/Characterization

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      <h1> <a alt="iGEM Nankai" class="ie6fixpic" title="iGEM Nankai" href="#">iGEM Nankai</a> </h1>
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<p><strong>Characterization</strong><br>
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   <strong>Plasmid and strain construction</strong><br>
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  What we have to do is insert the genes<em> rhlABRI</em> to <a name="OLE_LINK115"></a><a name="OLE_LINK114">the plasmid pBBR1MCS-2</a>. To accomplice this, we figure out 4 steps as followed.</p>
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              <ol>
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                <li>Restriction endonuclease site</li>
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              </ol>
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              <p>  Considering <em>P. aeruginosa</em> SQ6 is a wild-type strain, we retained the genes&rsquo; native promoter from the strain. Comparing the restriction sites of multiple cloning sites of the plasmid pBBR1MCS-2 and the genes<em> rhlABRI</em> including the promoter, we chose the restriction endonuclease sites, <em>Hind III</em> and <em>Sal I</em>.</p>
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              <ol>
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                <li>Premier designing</li>
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              </ol>
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              <p>Since it&rsquo;s hard to locate and identified the target genes <em>rhlABRI</em> using PCR in the whole-genome of <em>P. aeruginosa</em> SQ6, we compared six<em> rhlABRI</em> sequences from different <em>P. aeruginosa</em> strains, which were completed whole-genome sequencing in the database of NCBI. The GeneBank accession numbers are AE004091, CP000438, AP012280, HM190303, FM209186, and CP002496. On the basis of the terminal conserved regions of the six <em>rhlABRI</em> genes, we designed a pair of <em>rhl</em>ABRI PCR primers named rhl-F/R using the software <em>Oligo</em>. <br>
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                The forward primer rhl-F is 5'-GGTCT<u>GTCGAC</u>CGGTTTTTTCATGCCTT-3', with restriction endonuclease site of <em>Sal I</em> (the underlined nucleotide base), and the reverse primer rhl-R is 5'-GATGG<u>AAGCTT</u>GCAGAGAGACTACGCAA-GT-3', with restriction endonuclease site of <em>Hind III</em>.</p>
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              <ol>
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                <li>Recombinant cloning plasmid</li>
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              </ol>
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              <p>  We extracted the<em> P. aeruginosa</em> SQ6 genomic DNA using Bacterial Genomic DNA Miniprep Kit (AXYGEN, USA), and amplified the<em> rhlABRI</em>genes with their native promoters from <em>P. aerugnosa</em> SQ6 genomic DNA with the PCR primers rhl-F/R. The PCR system is as followed:</p>
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                <table border="1" cellspacing="0" cellpadding="0">
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                  <tr>
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                    <td width="187"><br>
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                      <em>P. aeruginosa</em> SQ6 genomic DNA </td>
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                    <td width="46"><p align="center">rhl-F</p></td>
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                    <td width="53"><p align="center">rhl-R</p></td>
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                    <td width="46"><p align="center">ddH2O</p></td>
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                    <td width="94"><p align="center">LA Taq enzyme</p></td>
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                  </tr>
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                  <tr>
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                    <td width="187"><p align="center">3μl</p></td>
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                    <td width="46"><p align="center">0.5μl</p></td>
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                    <td width="53"><p align="center">0.5μl</p></td>
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                    <td width="46"><p align="center">8.5μl</p></td>
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                    <td width="94"><p align="center">12.5μl</p></td>
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                  </tr>
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                </table>
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              </div>
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              <p>  But we failed the first time. Finally, we figured out it might because the genes<em> rhlABRI</em> is GC-rich sequence, so we add 6% DMSO in the PCR system to solve the problem. (DMSO can efficiently reduce the energy involved in the pairing of base G and base C.)<br>
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                  After cleaning up the <em>rhlABRI</em> PCR products and extracted the plasmid <a name="OLE_LINK117"></a><a name="OLE_LINK116">pBBR1MCS-2</a> from DH5α，we digested the genes <em>rhlABRI </em>and plasmid pBBR1MCS-2 with <em>Sal I </em>and <em>Hind III. </em>On the basis of digestion, we connect the genes <em>rhlABRI </em>and plasmid pBBR1MCS-2 with solution I to construct the recombinant expressing plasmid pBBR1-2rhl. <br>
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                DNA fragments and plasmids were purified with a PCR Cleanup Kit (AXYGEN, USA). DNA fragments from the gels were purified with a DNA Gel Extraction Kit (AXYGEN, USA). Plasmids in DH5α was extracted with a Plasmid Miniprep Kit (AXYGEN, USA).</p>
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              <ol>
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                <li>Transformation</li>
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              </ol>
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              <p>  The recombinant plasmids pBBR1-2rhl were transformed into E. coli DH5α by CaCl2–heat shock transformation. At first, added all connection product into the competent DH5α cell and flipped to mixture them. Then put it in the ice-bath for 0.5 hour, took it into the 42℃ water-bath for 1.5 minutes and put it back in the ice-bath for 2 minutes. After adding 800ul LB medium, cultured it for 1 hour in 37℃. Finally, took out 200ul to coated plates. After about 10 hours, monoclonal colony was selected to culture in LB medium. Finally, colony PCR was done to verify the plasmid. </p>
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              <p>Simulation Experiment in <em>E.coli</em> <br>
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                    We transformed pBBR1-2rhl, the constructed plasmid with <em>rhlABRI</em> genes, into <em>E.coli</em> <a name="OLE_LINK16"></a><a name="OLE_LINK15"></a><a name="OLE_LINK14">DH5α</a> by CaCl2-heat shocking transformation. To test the transformation result, we did the bacterial liquid PCR with the primer of <em>rhlR</em> gene. According to the result, the PCR product is around 900 bp, which is exactly the size of <em>rhlR</em> gene. This proves that the plasmid with<em> rhlABRI</em> gene had been successfully <a name="OLE_LINK289"></a><a name="OLE_LINK288">transfected </a>and amplified in<em> E.coli</em> DH5α<em>.</em><br>
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                <a name="OLE_LINK6"></a><a name="OLE_LINK5"></a><a name="OLE_LINK4"><img width="352" height="142" src="characterization_clip_image002.gif" alt="T-13 ABRI转化 成功 20141002"></a> <br>
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                    Next, we performed the<a name="OLE_LINK275"></a><a name="OLE_LINK274"></a><a name="OLE_LINK273"> high performance liquid chromatography (HPLC)</a> and thin layer chromatography (TLC) analysis to determine the component of the fermentation product. As is shown in the figure, the product has the same TLC result as purified rhamnolipid, and the HPLC results prove that the component of hydrolysis product is rhamose.<br>
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  <img width="335" height="275" src="characterization_clip_image004.gif" align="left" hspace="12" alt="QQ图片20141015204408.jpg">  <img width="122" height="273" src="characterization_clip_image006.gif" alt="C:\Users\Administrator\Desktop\o.jpg"> Then we characterize the property of the product to test the feasibility of our idea. We cultured 3 types of equivalent bacteria<em> E. coli</em> DH5α,<em> wildtype</em>, <a name="OLE_LINK8"></a><a name="OLE_LINK7"></a><a name="OLE_LINK3"><em>pBBR1</em></a><em>MCS-2</em> and <em>pBBR1-2rhl</em>, in the blank culture medium (7.5%NaNO3, 1%NaH2PO4, 1%K2HPO4, 0.1%Fe2(SO4)3, 0.1%Na2MoO4, 0.1%MgSO4, 0.1%CaCl2, 1%sucrose, 5%glycerinum) , and measured several parameters of each group respectively. According to the result, after 72 hours culture, the growth of <em>E.coli</em> DH5α carrying pBBR1-2rhl had no obvious difference from wild type and pBBR1MCS-2 empty vector, while the surface tension of its fermentation broth has significantly reduced, and the emulsifying ability has remarkably improved.</p>
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              <table border="1" cellspacing="0" cellpadding="0" width="503">
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                  <td width="210" valign="top"><br>
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                    <a name="_Hlk401094260">Liquid </a></td>
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                  <td width="92" valign="top"><p>Bacteria growth</p></td>
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                  <td width="99" valign="top"><p>Surface tension</p></td>
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                  <td width="103" valign="top"><p>Emulsifying ability</p></td>
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                </tr>
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                <tr>
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                  <td width="210" valign="top"><p>Pure culture medium</p></td>
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                  <td width="92" valign="top"><p>-</p></td>
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                  <td width="99" valign="top"><p>68.20</p></td>
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                  <td width="103" valign="top"><p>-</p></td>
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                </tr>
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                <tr>
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                  <td width="210" valign="top"><p>E.coli DH5<a name="OLE_LINK11"></a><a name="OLE_LINK10"></a><a name="OLE_LINK9">α</a> wildtype</p></td>
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                  <td width="92" valign="top"><p>+++</p></td>
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                  <td width="99" valign="top"><p>63.75</p></td>
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                  <td width="103" valign="top"><p>-</p></td>
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                </tr>
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                <tr>
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                  <td width="210" valign="top"><p>E.coli DH5α with pBBR1MCS-2 empty vector</p></td>
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                  <td width="92" valign="top"><p>+++</p></td>
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                  <td width="99" valign="top"><p>65.24</p></td>
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                  <td width="103" valign="top"><p>-</p></td>
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                </tr>
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                <tr>
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                  <td width="210" valign="top"><p>E.coli DH5<a name="OLE_LINK272"></a><a name="OLE_LINK271"></a><a name="OLE_LINK270">α</a> with pBBR1MCS-2rhl</p></td>
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                  <td width="92" valign="top"><p>+++</p></td>
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                  <td width="99" valign="top"><p>39.34</p></td>
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                  <td width="103" valign="top"><p>+</p></td>
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                </tr>
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              </table>
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              <p>* The number of &ldquo;+&rdquo; reflects the degree of each parameters.<br>
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                <strong>Transformation to<em> Pseudomonas stutzeri</em></strong><br>
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                Transformation method discussion<br>
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                At first we used CaCl2-heat shock transformation to transform plasmid pBBR1<a name="OLE_LINK256"></a><a name="OLE_LINK255">-2rhl</a> into P. stutzeri 10-5, then switched to electroporation because of the low transformation efficiency of the P. stutzeri 10-5 competent cells we made by our own. Although electroporation is generally believed to have a higher transformation efficiency than CaCl2-heat shock transformation, we did not achieve our desired results. According to bacteria liquid PCR result, no <em>rhlABRI</em> copies were found in the culture mixture.<br>
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                *We used electric shock buffer solution and sucrose for two different electroporation protocols, respectively. See Methods below.<br>
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                Different plasmid structures influence transformation efficiency<br>
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                The failure of both chemical transformation and electroporation leads to the discussion of <a name="OLE_LINK2"></a><a name="OLE_LINK1">pBBR1MCS-2</a>, the plasmid backbone that we used in construction of pBBR1-2rhl. Although pBBR1MCS-2 is a high copy and broad host range vector, its great length (5144bp) could affect the transformation efficiency, especially when recombined with the 4407bp <em>rhlABRI</em>. Meanwhile, P. stutzeri 10-5 is an wild strain newly acquired from an natural oil reservoir. Thus, without any genetical modification, P. stutzeri 10-5 may not fit the 9524bp pBBR1-2rhl easily.<br>
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                So we choose two smaller plasmid, pSB1C3 (with chloramphenicol resistance) and pUCP20 (with carbenicillin resistance), for substitute plasmid backbones. To test whether this two plasmids are suitable for P. Stutzeri transformation, two plasmid containing reporter genes, pSB1C3-mRFP1 (BBa_J04450) and pUCP20-mapple, was transformed into P. stutzeri 10-5 via sucrose electroporation, respectively. <br>
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                pSB1C3-mRFP1 is a standard plasmid provided by igem.org to test transformation efficiency of competent cells, while pUCP20-mapple is a recombinant plasmids derived from pUC20 plasmid and is inserted with mapple, an engineered red fluorescence protein coding gene. The reason of choosing pUCP20-mapple for testing is that previous work with<em> P. aeruginosa</em> in our lab had been carried successfully using it. Taking account of the genetic similarities between<em> P.aeruginosa</em> and <em>P.stutzeri</em>, we assume pUCP20-mapple functions in <em>P.stutzeri</em> as well.<br>
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                <br>
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                (pSB1C3-mRFP1)</p>
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              <p align="center">(pUCP20-mapple)<br>
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                According to the result, pSB1C3-mRFP1 transformed bacteria formed colonies on LB agar plate containing chloramphenicol, but the colonies are not visibly red under UV. This result indicated that the plasmid with CamR was possibly transformed into the recipient bacteria, but the promoter of RFP can not be activated. Thus, when constructing <em>rhlABRI</em> with pSB1C3 backbone, we need to select different promoter for the gene to be expressed. In the mean time, pUCP20-mapple transformed bacteria formed colonies on LB agar plate containing carbenicillin as well and the colonies are visibly red under UV, indicating that replacing mapple coding gene with <em>rhlABRI</em> in pUCP20 plasmid can be a possible way to build an expression plasmid suitable for<em> P. stutzeri</em>.<br>
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                Due to limited time, we just finished constructing the pUCP20-rhlABRI plasmid and transformed it into <em>P. stutzeri</em> 10-5 via sucrose electroporation once. Unfortunately, the colonies appeared on LB agar plate containing carbenicillin can not grow in liquid LB containing carbenicillin when picked up, indicating the transformed bacteria are not in good condition.</p>
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              <p><strong>Methods</strong><br>
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                <strong><em>Experiments on E.coli</em></strong><strong> </strong><br>
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                <strong>1. Preparation and transformation of competent cells</strong><br>
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                Pick activated <em>E.coli</em> DH5α single colony from the LB plate and add it into 5ml LB liquid medium at the temperature of 37℃ about 12 hours. Inoculated the bacterial in 100ml LB liquid medium at the ratio of 1:100(37℃for 2-3 hours),until its OD(ƛ=600) = 0.5 <br>
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                The medium is transferred to a centrifuge tube on ice, placed 10 minutes, and then to <a name="OLE_LINK283"></a><a name="OLE_LINK282">4</a>℃, 3000rpm,centrifuge for 10 minutes </p>
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              <p>Discard the supernatant, gently suspense cells by precooling 0.05mol/L CaCl2 10ml, ice placed for 15-30 minutes. 4℃,3000rpm centrifuge for 10 minutes. Repeat three times. <br>
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                Discard the supernatant, add 4ml precooling 0.05mol/L CaCl2(containing 15% glycerol), gently suspense cells, put them on the ice for a few minutes.<br>
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                Pack competent cells 100μl into every tube, store them in -70 C.<br>
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                Add recombinant plasmid into competent cells, ice bath 30min.<br>
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                42℃, heat shock 90s. <br>
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                Ice bath 2-3min.<br>
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                Add 800μl preheat（37℃) recovery medium, shaking 45min(37℃，200rpm).<br>
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                5000rpm centrifuge 3min, discard supernatant, <a name="OLE_LINK262"></a><a name="OLE_LINK261">coat </a>plate after suspending.<br>
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                Cultivate overnight, 37℃, inverted.<br>
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   <strong>2. <a name="OLE_LINK30"></a><a name="OLE_LINK268"></a><a name="OLE_LINK267">Thin layer chromatography</a> (TLC)</strong><strong> </strong><br>
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                The product was separated, visualized and compared with the purified rhamnolipid by <a name="OLE_LINK222">thin layer chromatography (TLC) on Silica gel G plates</a>. A 3μL sample was placed on the Silica gel G plates. After drying at room temperature, the chromatograms were developed with chloroform-methanol-water and visualized with a <a name="OLE_LINK87"></a><a href="file://localhost/dict/::key.0895DFE8DB67F9409DB285590D870EDD:sulfuric%2520acid-phenol" title="查找: sulfuric acid-phenol">sulfuric acid-phenol</a> TLC reagent at 110 °C for 5&nbsp;min.<br>
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  <strong>3. High Performance Liquid Chromatography (HPLC)</strong><br>
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  <a name="OLE_LINK279"></a><a name="OLE_LINK278">    Add appropriate amount of </a><a href="http://cnc.dict-client.iciba.com/2013-01-22/?action=client&word=%E4%B8%89%E6%B0%9F%E4%B9%99%E9%85%B8&dictlist=201,2,1,101,6,104,7,105,5,103,203,202,8,9,204,205,10,11,3,4,&zyid=%E7%89%A9%E7%90%86%E5%AD%A6&hyzonghe_tag=2&nav_status=1&type=0&authkey=abb5d01d6ff6796844ec7d37f28f3b08&uuid=4758B9E3794342E1B46A3735C4DAD583&uid=9999624&v=2014.05.16.044&tip_show=2,1,3,4,5,6,&fontsize=0&channel=21.00###">trifluoroacetic acid </a> (2 mol/L) into the purified product, 110℃, hydrolyze for 8h. <br>
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                Cool to room temperature , 1000rpm, centrifuge for 5 minutes.<br>
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                Add 0.3 mol/L NaOH into supernatant for neutralization, adjust PH to 7.0. <br>
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                    Add 10ul sample of standard monosaccharide and hydrolysis product for measurement. <br>
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                (Mobile phase: ultrapure water; column temperature: RT; velocity:0.5ml/min)</p>
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              <p><strong><em>Pseudomonas stutzeri</em></strong><strong> electroporation (using electric shock buffer solution)</strong></p>
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              <ol>
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                <li><strong>Preparation of competent<em> P.stutzeri</em> cells</strong></li>
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                <li>Select <em>P.stutzeri</em> monoclonal colony in 5ml LB medium, 35°C, 180 rpm;</li>
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                <li>After about 12 hours, a 1 ml <em>P.stutzeri</em> seed culture was inoculated in a 100 ml LB medium and then incubated at 35°C, 250 rpm until the OD600 ratio is about 0.6; (culture it in 35°C is to inactivate the restriction modification system)</li>
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                <li>Put the <em>P.stutzeri</em> LB medium in ice-bath for 20 minutes, and spin at 4℃，5000rpm, 5 minutes with 50ml centrifugal tube; </li>
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                <li>Discard the upper phase, add 50ml electric shock buffer solution (incude 15％glycerol，1mM MOPS) and mix well, and spin at 4℃，5000rpm, 10 minutes;</li>
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                <li>Discard the upper phase, add 50ml electric shock buffer solution and mix well, and spin at 4℃，5000rpm, 10 minutes; Repeat this step for three times;</li>
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                <li>Discard the upper phase, add 800ul electric shock buffer solution and mix well, and add 100ul to each 1.5ml tube.</li>
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                <li>Use it in electroporation immediately, or the efficiency will be low after store it in fridge, 80℃. </li>
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                <li><strong>Electroporation</strong></li>
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                <li>Add 3ul plasmid pBBR1-2rhl into the 100ul competent<em> P.stutzeri</em> cells, flip to mix well. Transfer all the cells into a cold 2mm diameter electroporation-cup, shock it in the electric field at 3.5kV, for about 5.0mS. </li>
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                <li>Add 800ul SOC medium immediately, mix well and transfer them into a 1.5ml tube. Culture the cells for 3 hours at 35℃, 180rpm. Transfer 200ul to coated LB plates (with 25ug/ml kanamycin). Invert the plateand put it in thermotank at 35℃ overnight.</li>
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                <li>Change the thermotank&rsquo;s temperature to 30℃ for 24 to 48 hours.</li>
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                <li>Select monoclonal colony to culture in LB medium. Colony PCR was done to verify the plasmid at first, and then extracted the plasmid and digested it with <em>Sal</em> I and <em>Hind</em> III to see if the plasmid transformed is right.</li>
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              </ol>
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              <p><strong><em>Pseudomonas stutzeri</em></strong><strong> electroporation (using sucrose)</strong><br>
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                <strong>1. </strong><strong>Preparation of competent<em> P.stutzeri</em> cells</strong><br>
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                a. Select P.stutzeri monoclonal colony in 4ml LB medium, 30°C, 200 rpm;<br>
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                b. After about 24 hours, spin at 16000g, 2 minutes with 2ml centrifugal tube, discard the upper phase, add the other 2ml, spin again, discard the upper phase; <br>
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                c. Resuspend with 500μl 300mM sucrose, spin at 16000g, 2 minutes, discard the upper phase, do this 3 times;<br>
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                d. Resuspend with 100μl 300mM sucrose.<br>
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  <strong>2. Electroporation</strong><br>
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                a. Add 50ng plasmid into the 50ul competent P.stutzeri cells, flip to mix well. Transfer all the cells into a cold 2mm diameter electroporation-cup, shock it in the electric field at 3.5kV, for about 5.0mS. <br>
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                b. Add 1ml LB medium immediately, mix well and transfer them into a 1.5ml tube. Culture the cells for 1 hours at 30℃, 180rpm. <br>
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                c. Transfer 100ul to coated LB plates containing corresponding antibiotic. <br>
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                d. Invert the plateand put it in thermotank at 30℃ overnight.<br>
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                e. Select monoclonal colony to culture in LB medium containing corresponding antibiotic. </p>
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