|
|
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| <ul class="tabs tabs-wrapper transparent"> | | <ul class="tabs tabs-wrapper transparent"> |
| <li class="tab col l2 m2 s2"> | | <li class="tab col l2 m2 s2"> |
− | <a href="#Overview" class="blue-text active waves-effect waves-light">Overview</a> | + | <a href="#Basic_Parts" class="blue-text active waves-effect waves-light">Basic Parts</a> |
| </li> | | </li> |
| <li class="tab col l2 m2 s2"> | | <li class="tab col l2 m2 s2"> |
− | <a href="#Chemotaxis_Modification" class="blue-text waves-effect waves-light">Chemotaxis | + | <a href="#Composite_Parts" class="blue-text waves-effect waves-light">Composite Parts</a> |
− | Modification</a>
| + | |
− | </li>
| + | |
− | <li class="tab col l2 m2 s2">
| + | |
− | <a href="#Permeability_Improvement"
| + | |
− | class="blue-text waves-effect waves-light">Permeability Improvement</a>
| + | |
− | </li>
| + | |
− | <li class="tab col l2 m2 s2">
| + | |
− | <a href="#Adhesion_Assay" class="blue-text waves-effect waves-light">Adhesion Assay</a>
| + | |
| </li> | | </li> |
| <li class="tab col l2 m2 s2"> | | <li class="tab col l2 m2 s2"> |
| <a href="#Parts" class="blue-text waves-effect waves-light">Parts</a> | | <a href="#Parts" class="blue-text waves-effect waves-light">Parts</a> |
| </li> | | </li> |
| + | |
| </ul> | | </ul> |
| </div> | | </div> |
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| <li><a href="https://2015.igem.org/Team:USTC" class="waves-effect waves-light">Home</a></li> | | <li><a href="https://2015.igem.org/Team:USTC" class="waves-effect waves-light">Home</a></li> |
| <li><a href="https://2015.igem.org/Team:USTC/Description" class="waves-effect waves-light">Project</a></li> | | <li><a href="https://2015.igem.org/Team:USTC/Description" class="waves-effect waves-light">Project</a></li> |
− | <li><a href="https://2015.igem.org/Team:USTC/Modeling" class="waves-effect waves-light">Modeling</a></li>
| |
| <li><a href="https://2015.igem.org/Team:USTC/Achievements" class="waves-effect waves-light">Achivements</a></li> | | <li><a href="https://2015.igem.org/Team:USTC/Achievements" class="waves-effect waves-light">Achivements</a></li> |
| <li><a href="https://2015.igem.org/Team:USTC/Tutorials" class="waves-effect waves-light">Tutorials</a></li> | | <li><a href="https://2015.igem.org/Team:USTC/Tutorials" class="waves-effect waves-light">Tutorials</a></li> |
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| <div class="row"> | | <div class="row"> |
| <div class="col offset-m1 offset-l2 s12 m10 l8"> | | <div class="col offset-m1 offset-l2 s12 m10 l8"> |
− | <div id="Overview" class="row"> | + | <div id="Basic_Parts" class="row"> |
| <div class="card hoverable"> | | <div class="card hoverable"> |
| <div class="col s12"> | | <div class="col s12"> |
| <div class="card-content"> | | <div class="card-content"> |
− | <p>That is so amazing! We finally made NDM this year! The results page will deliver the fresh | + | <p>This year, we choose several parts to conduct our projects. The main functions of the |
− | results from us. Our results include these sections:</p> | + | function we use are as follow:</p> |
− | <ul> | + | <ol> |
− | <li><p>Permeability Improvement: In this section, we will demonstrate our synthetic | + | <li>Promoters.</li> |
− | bioloical base constrcuction, characterization of permeability improvement. This is the | + | <li>Proteins to improve the bactial permeability.</li> |
− | fundation of CACCI construction, as well as NDM.</p> | + | <li>Porins.</li> |
− | </li> | + | <li>Proteins to improve the mobile of bacteria.</li> |
− | <li><p>Chemotaxis Modification: This section introduces results of chemotaxis | + | <li>Sense system.</li> |
− | engineering.</p> | + | </ol> |
− | </li> | + | <table class="striped"> |
− | <li><p>Adhesion Assay: This section explicitly explain adhesion methods, adhesion dynamics | + | <thead> |
− | and adhesion protocols recommended for all users, which is an important part of our | + | <tr> |
− | results.</p> | + | <th>Code Name</th> |
− | </li> | + | <th>Part Name</th> |
− | <li><p>Antibiotics Concentration Detection Demo: This section includes our initial test on | + | <th>Function</th> |
− | our system, how we define our final methods and how we establshed protocols for | + | </tr> |
− | users.</p> | + | </thead> |
− | </li> | + | <tbody> |
− | <li><p>Calibration</p> | + | <tr> |
− | </li>
| + | <td><a href="http://parts.igem.org/Part:BBa_K1593207">BBa_K1593207</a></td> |
− | </ul>
| + | <td>micF</td> |
− | <p>Want to get them now? Click their title directly!</p>
| + | <td>Promoter sensing sulfoamid</td> |
| + | </tr> |
| + | <tr> |
| + | <td><a href="http://parts.igem.org/Part:BBa_K1593208">BBa_K1593208</a></td> |
| + | <td>SoxS</td> |
| + | <td>Promoter sensing tetracycline</td> |
| + | </tr> |
| + | <tr> |
| + | <td><a href="http://parts.igem.org/Part:BBa_K1593209">BBa_K1593209</a></td> |
| + | <td>OprF</td> |
| + | <td>The major porin of Pseudomonas aeruginosa allowing diffusion of polysaccharides in a |
| + | range of 2000 to 3000 Da. |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td><a href="http://parts.igem.org/Part:BBa_K1593666">BBa_K1593666</a></td> |
| + | <td>SCVE</td> |
| + | <td>SARS Coronavirus Envelope Protein, improving the bactial permeability.</td> |
| + | </tr> |
| + | <tr> |
| + | <td><a href="http://parts.igem.org/Part:BBa_K1593998">BBa_K1593998</a></td> |
| + | <td>cheZ</td> |
| + | <td>Chemotactic protein. When it is overexpressed, the mobility of bacteria will be |
| + | improved. |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td><a href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a></td> |
| + | <td>T7-RBS-SCVE</td> |
| + | <td>Circuit containing strong expression of SCVE regulated by T7, which will |
| + | significantly improve bacterial permeability. |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td><a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a></td> |
| + | <td>T7-RBS-OprF</td> |
| + | <td>Using T7 strong promoter to express OprF, derived from Pseudomonus areuginosa, would |
| + | strongly improve the bacterial permeability. |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td><a href="http://parts.igem.org/Part:BBa_K1593997">BBa_K1593997</a></td> |
| + | <td>R0010-RBS-cheZ</td> |
| + | <td>Used to coding a chemotactci protein, cheZ, downstream of lac promotor.</td> |
| + | </tr> |
| + | <tr> |
| + | <td><a href="http://parts.igem.org/Part:BBa_K1593211">BBa_K1593211</a></td> |
| + | <td>micF-RBS-GFP</td> |
| + | <td>A basic part on antibiotic substance sensing-reporting system.</td> |
| + | </tr> |
| + | <tr> |
| + | <td><a href="http://parts.igem.org/Part:BBa_K1593001">BBa_K1593001</a></td> |
| + | <td>COMPX</td> |
| + | <td>Express an outmembrane protein with a point mutation</td> |
| + | </tr> |
| + | </tbody> |
| + | </table> |
| | | |
| </div> | | </div> |
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Line 126: |
| | | |
| | | |
− | <div id="Chemotaxis_Modification" class="row"> | + | <div id="Composite_Parts" class="row"> |
| <div class="card hoverable"> | | <div class="card hoverable"> |
− | <div class="row"> | + | <div class="col s12"> |
− | <div class="col s12"> | + | <div class="card-content"> |
− | <div class="card-content"> | + | <p>A list of composit parts USTC iGEM 2015 have submitted to the Registry.The main functions of |
− | <p><strong>Growth characteristics</strong><br>We detected OD600(nm) to identify the effect | + | these composite parts are as following:</p> |
− | of cheZ expression on <em>E. coli</em> through time.<br><img | + | <ol> |
− | src="https://static.igem.org/mediawiki/2015/d/d6/Ustc-growth.png" alt="图片名称"></p>
| + | <li>To improve the permeability of bacteria;</li> |
| + | <li>To modify the chemotaxis of bacteria;</li> |
| + | <li>To modify outmembrane protein;</li> |
| + | <li>To test the response of promtor.</li> |
| + | </ol> |
| + | <table class="striped"> |
| + | <thead> |
| + | <tr> |
| + | <th>Code Name</th> |
| + | <th>Parts Name</th> |
| + | <th>Type</th> |
| + | <th>Function</th> |
| + | </tr> |
| + | </thead> |
| + | <tbody> |
| + | <tr> |
| + | <td><a href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a></td> |
| + | <td>T7-RBS-SCVE</td> |
| + | <td>Translational_Unit</td> |
| + | <td>overexpress SCVE to improve the permeability of bacterial</td> |
| + | </tr> |
| + | <tr> |
| + | <td><a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a></td> |
| + | <td>T7-RBS-OprF</td> |
| + | <td>Coding</td> |
| + | <td>overexpress OprF to improve the permeability of bacterial</td> |
| + | </tr> |
| + | <tr> |
| + | <td><a href="http://parts.igem.org/Part:BBa_K1593997">BBa_K1593997</a></td> |
| + | <td>R0010-RBS-cheZ</td> |
| + | <td>Coding</td> |
| + | <td>overexpress cheZ under the control of lac to improve the chemotaxis of bacterial |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td><a href="http://parts.igem.org/Part:BBa_K1593211">BBa_K1593211</a></td> |
| + | <td>micF-RBS-GFP</td> |
| + | <td>Translational_Unit</td> |
| + | <td>test the response of micF to antibiotics using the GFP report system</td> |
| + | </tr> |
| + | <tr> |
| + | <td> |
| + | <a href="http://parts.igem.org/cgi/partsdb/part_info.cgi?part_id=38524">BBa_K1593888</a> |
| + | </td> |
| + | <td>tRNA&tRNA Synthetase</td> |
| + | <td>Translational_Unit</td> |
| + | <td>express a rare amino acid,pAzF</td> |
| + | </tr> |
| + | <tr> |
| + | <td><a href="http://parts.igem.org/Part:BBa_K1593002">BBa_K1593002</a></td> |
| + | <td>tRNA complex with Compx</td> |
| + | <td>Device</td> |
| + | <td>orthogonal system to express Compx</td> |
| + | </tr> |
| + | <tr> |
| + | <td><a href="http://parts.igem.org/Part:BBa_K1593000">BBa_K1593000</a></td> |
| + | <td>lac-compx with RFP</td> |
| + | <td>Device</td> |
| + | <td>overexpress compx and detect its quantity by GFP</td> |
| + | </tr> |
| + | </tbody> |
| + | </table> |
| | | |
− | <p>The exogenous protein expression affects the growth of bacteria, which was indicated by
| |
− | slight slower grow-up in <em>E. coli</em> containing cheZ plasmid. In total, cheZ
| |
− | doesn't have obvious negative impact on bacteria grown. </p>
| |
− |
| |
− | <p><strong>SDS-PAGE</strong><br>After induced by IPTG in 1mM about 4 h, cheZ was found in
| |
− | SDS-PAGE gel, compared to the wild type (Top10) and bacteria without IPTG induction.
| |
− | Through this result, we concluded that cheZ has been successfully expressed with the
| |
− | IPTG induction.<br><img
| |
− | src="https://static.igem.org/mediawiki/2015/7/72/Ustc-chez_expression.png" alt="图片名称"><br>See
| |
− | protocol in <a href="http://tower.im">Protocols: SDS-PAGE</a></p>
| |
− | </div>
| |
| </div> | | </div> |
| </div> | | </div> |
− | </div>
| |
− | </div>
| |
| | | |
− | <div id="Permeability_Improvement" class="row">
| |
− | <div class="card hoverable">
| |
− | <div class="row">
| |
− | <div class="col s12">
| |
− | <div class="card-content">
| |
− | <h4 id="Extraction_of_Permeability_Improvement_Fragements" class="scrollspy">Extraction of
| |
− | Permeability Improvement Fragements</h4>
| |
− |
| |
− | <p>We successfully get
| |
− | gene fragements that are used for improving bacterial permeability, which including,
| |
− | </p>
| |
− | <ul>
| |
− | <li>SCVE, a viroporin originally from SARS virus, is synthesized by Sangon Co. ctd.</li>
| |
− | </ul>
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/1/1d/Ustc-scve.jpeg"
| |
− | alt="Figure 1: Extraction of SCVE fragement"></p>
| |
− | <ul>
| |
− | <li>OprF, a bigger porin compared with OmpF in <em>E. coli</em>, is extracted from <em>P.aeruginosa,
| |
− | PAO1</em> genome by PCR.
| |
− | </li>
| |
− | </ul>
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/6/6d/Ustc-t7oprf_lac-chez.jpeg"
| |
− | alt="Figure 2: Extraction of OprF fragement"></p>
| |
− | <ul>
| |
− | <li><p>T7 is a strong promoter which we got form Parts Registry. T7 will be used to
| |
− | trigger the expression of both OprF and SCVE.<br><img
| |
− | src="https://static.igem.org/mediawiki/2015/7/7a/Ustc-T7.jpeg"
| |
− | alt="Figure 3: T7 extraction from parts"></p>
| |
− | </li>
| |
− | <li><p>Cas9 is got from 2015 Part Distribution.</p>
| |
− | </li>
| |
− | </ul>
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/1/1c/USTC-Cas9.png"
| |
− | alt="Figure 4: Extraction of Cas9 from parts"></p>
| |
− | <ul>
| |
− | <li>gRNA-AcrB and gRNA-EmrE, these two fragments are for silencing bacterial
| |
− | transmembrane protein AcrB and EmrE to strongly block drug efflux system. These two
| |
− | parts are synthesized by Sangon Biotech Company.
| |
− | </li>
| |
− | </ul>
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/5/54/USTC-gRNA_extraction.png"
| |
− | alt="Figure 5: Extraction of gRNA-AceB and gRNA-EmrE, two fragments are synthesized by Sangon Biotech Company">
| |
− | </p>
| |
− |
| |
− | <h4 id="Construction_of_Permeability_Improving_Plasmid" class="scrollspy">Construction of
| |
− | Permeability Improving Plasmid</h4>
| |
− | <ul>
| |
− | <li>Construction of plasimid with T7-SCVE (<a
| |
− | href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>) . We finally
| |
− | ligated the T7 with SCVE and got T7-SCVE (<a
| |
− | href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>).
| |
− | </li>
| |
− | </ul>
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/e/ed/Ustc-scve_jp_t7.jpeg"
| |
− | alt="Figure 6: Construction of T7-SCVE ([BBa_K1593667](http://parts.igem.org/Part:BBa_K1593667))">
| |
− | </p>
| |
− | <ul>
| |
− | <li>Construction of plasimid containing T7-OprF (<a
| |
− | href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>). This is the
| |
− | result of T7 with OprF.
| |
− | </li>
| |
− | </ul>
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/6/6d/Ustc-t7oprf_lac-chez.jpeg"
| |
− | alt="Figure 7: Construction of T7-OprF ([BBa_K1593210](http://parts.igem.org/Part:BBa_K1593210))">
| |
− | </p>
| |
− |
| |
− | <h4 id="Growth_Characterization" class="scrollspy">Growth Characterization</h4>
| |
− |
| |
− | <p>We firstly characterized the growth rate of genetically modified CACCI along with wild
| |
− | type BL21 using optical density at 600 nm, which is called OD 600, in order to
| |
− | demonstrate that the overexpression of porin(OprF with T7,<a href="http://tower.im">BBa_K1593209</a>)
| |
− | or viroporin(SCVE with T7, (<a href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>)
| |
− | won't severely affect the growth of bacteria, at least it won't significantly inhibit
| |
− | bacteria growth and become a kill switch.</p>
| |
− |
| |
− | <p>All bacteria are cultured previously in LB medium about 12 h. And then the measurement of
| |
− | bacteria growth through time began at 8 A.M. the other day.</p>
| |
− |
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/d/d8/Ustc-growth2.png"
| |
− | alt="Figure 8: Bacteria growth characterization"></p>
| |
− |
| |
− | <p>As the images above illustrated, we found <em>E. coli</em> BL21 wild type has the best
| |
− | growth characteristics with a maximal OD600=3.427. Whereas <em>E. coli</em> BL21 with
| |
− | OprF (<a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>) has a
| |
− | relatively low optical density as its OD600 after 12 h is 2.808 compared to the
| |
− | wildtype. In the same way, characterization of <em>E. coli</em> BL21 with SCVE, <a
| |
− | href="http://parts.igem.org/Part:BBa_K1593667">http://parts.igem.org/Part:BBa_K1593667</a>
| |
− | showed a slightly smaller OD600, as its finally turned to 2.835. <em>E. coli</em> BL21
| |
− | with T7-OprF (<a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>) and
| |
− | <em>E. coli</em> BL21 with T7-SCVE, (<a href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>)
| |
− | are showing 19% decreased maximal cell density and 18.5% decreased maximal cell density
| |
− | respectively in comparison with wild type.</p>
| |
− |
| |
− | <p>Consequently, the result implies that the genetical modification on bacterial
| |
− | permeability, at least the overexpression of OprF and SCVE with a strong promoter T7,
| |
− | won't significantly influence the bacterial growth. Thus, genetically modified bacteria
| |
− | can be used in the following experiments.</p>
| |
− |
| |
− | <p>Later on, we will characterize the basic permeability capability of <em>E. coli</em> BL21
| |
− | with T7-OprF (<a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>) and
| |
− | <em>E. coli</em> BL21 with T7-SCVE, (<a href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>).
| |
− | </p>
| |
− |
| |
− | <h4 id="ONPG_Assay" class="scrollspy">ONPG Assay</h4>
| |
− |
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/5/54/ONPG-ustc-sche.png"
| |
− | alt="Figure 9: ONPG reaction"></p>
| |
− |
| |
− | <p>ONPG, that is the abbreviation of ortho-Nitrophenyl-β-galactoside. ONPG is a colorimetric
| |
− | and spectrophotometric substrate, previously used for detecting β-galactosidase
| |
− | activity. ONPG is actually colorless at normal situation, however if it triggers the
| |
− | degradation reaction catalyzed by β-galactosidase, we will get galactose and
| |
− | ortho-nitrophenol. Surprisingly, the compound ortho-nitrophenol has a yellow color,
| |
− | which means we are able to detect the chemical reaction through OD detection. Besides,
| |
− | owing to the existence of β-galactosidase in bacteria, a more intense optical density we
| |
− | detected, that means there are more ONPG uptaked by bacteria. Therefore, using ONPG we
| |
− | are able to distinguish the permeability capability of different bacteria.</p>
| |
− |
| |
− | <p>Here's our result.</p>
| |
− |
| |
− | <p>Note: The wavelength for detecting the bacterial concentration and absortion of ONPG are
| |
− | respectively 600nm and 406nm.</p>
| |
− |
| |
− | <p>First, we detected the bacterial solutions to control the initial concentrations. </p>
| |
− |
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/3/32/Ustc-board.png"
| |
− | alt="Figure 10: ONPG assay schematic show"></p>
| |
− |
| |
− | <p>The OD values for <em>E.coli </em>BL21, <em>E.coli </em>BL21 with T7-OprF (<a
| |
− | href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>) , <em>E.coli</em>
| |
− | BL21 with T7-SCVE(<a href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>)
| |
− | are respectively 2.856,1.806 and 1.889. Based on these, we know that the concentrations
| |
− | of these three bacteria are close.</p>
| |
− |
| |
− | <p>Then we add ONPG to the bacterial solutions and detect the OD.</p>
| |
− |
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/5/52/Ustc-onpg88.png"
| |
− | alt="Figure 11: ONPG assay, absorption OD406"></p>
| |
− |
| |
− | <p>From this figure, we can see a rising trend in all bacterial solutions. The OD values for
| |
− | <em>E.coli</em> BL21 with T7-OprF(<a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>)
| |
− | and <em>E.coli </em>BL21 with T7-SCVE(<a href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>)
| |
− | are respectively 0.2520 and 0.2588,both are higher than <em>E.coli</em> BL21,the wild
| |
− | type. We can infer that the absorbtion of modified bacteria is higher than the wild
| |
− | type.</p>
| |
− |
| |
− | <p>There is a more visual figure for comparing the absorbtions. We made a zero calibration
| |
− | using the OD value of wild type as the zero level.</p>
| |
− |
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/0/00/Ustc-onpg55.png"
| |
− | alt="Figure 12: ONPG assay, absorption OD406 in average"></p>
| |
− |
| |
− | <p>In general, the OD values of both two modified bacteria are rising. For<em> E.coli</em>
| |
− | BL21 with T7-OprF(<a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>),
| |
− | the rising is relatively smooth. For <em>E.coli</em> BL21 with T7-SCVE(<a
| |
− | href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>),the value goes
| |
− | down first, then goes up .And the <em>E.coli</em> BL21 with T7-SCVE(<a
| |
− | href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>) has a higher
| |
− | absorbtion than <em>E.coli </em>BL21 with T7-OprF(<a
| |
− | href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>).</p>
| |
− |
| |
− | <h4 id="NPN_Assay" class="scrollspy">NPN Assay</h4>
| |
− |
| |
− | <p><img
| |
− | src="https://static.igem.org/mediawiki/2015/1/16/Ustc-npn.gif"
| |
− | alt="Figure 13: Schematic Structure of N-Phenyl-1-naphthylamine, NPN"></p>
| |
− |
| |
− | <p>N-Phenyl-1-naphthylamine(NPN),C16H13N, CAS:90-30-2, FW: 219.29, is a nonpolar probe,
| |
− | whose fluorescence signal is relatively strong in a phospholipid environment, but weak
| |
− | in aqueous surroundings. Consequently, using this feature, we are able to characterize
| |
− | bacteria permeability by measuring the fluorescence intensity of the bacteria solution.
| |
− | When NPN molecules are absorbed by bacteria, we will observe a significantly rising
| |
− | fluorescence intensity. The more fluorescence intensity we got, the more permeable the
| |
− | bacteria are. Here are what we got from NPN uptake assay and the conclusion on
| |
− | genetically engineered bacteria permeability improvement. </p>
| |
− |
| |
− | <p>Here's our results, the first picture illustrate the total fluorescence intensity
| |
− | measurement by <a href="http://www.bmglabtech.com/en/products/clariostar/">BMG Labtech
| |
− | CLARIOstar®</a>.</p>
| |
− |
| |
− | <p><em>Note</em>: The fluorescence intensity signal at excitation wavelength range is 305nm
| |
− | to 335nm and emission wavelength range is 370nm to 410nm, which is recommended by
| |
− | previous research. </p>
| |
− |
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/e/e5/Ustc-npn1.png"
| |
− | alt="Figure 14: NPN uptake result-1"></p>
| |
− |
| |
− | <p>The total fluorescence intensity of <em>E. coli</em> BL21 wild type is 126451. On the
| |
− | other hand, the fluorescence intensity in <em>E. coli</em> BL21 with T7-OprF (<a
| |
− | href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>) and <em>E.
| |
− | coli</em> BL21 with T7-SCVE, (<a href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>)
| |
− | are respectively 207322 and 151820, which are significantly larger than the wildtype
| |
− | assay.</p>
| |
− |
| |
− | <p>Though we have already got the exact data on NPN uptake, we still need to revise its
| |
− | effectiveness because of the different bacteria concentration of <em>E. coli</em> BL21
| |
− | wild type, <em>E. coli</em> BL21 with T7-OprF (<a
| |
− | href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>) and <em>E.
| |
− | coli</em> BL21 with T7-SCVE, (<a href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>).
| |
− | Here is the exact bacteria concentration data, based on OD600nm,</p>
| |
− | <table>
| |
− | <thead>
| |
− | <tr>
| |
− | <th>Strain</th>
| |
− | <th>OD600nm</th>
| |
− | </tr>
| |
− | </thead>
| |
− | <tbody>
| |
− | <tr>
| |
− | <td><em>E. coli</em> BL21 wild type</td>
| |
− | <td>3.663</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td><em>E. coli</em> BL21 with T7-OprF (<a
| |
− | href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>)
| |
− | </td>
| |
− | <td>2.431</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td><em>E. coli</em> BL21 with T7-SCVE, (<a
| |
− | href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>)
| |
− | </td>
| |
− | <td>2.441</td>
| |
− | </tr>
| |
− | </tbody>
| |
− | </table>
| |
− | <p>After correcting these data, we are able to get bacteria absorption uptake capability in
| |
− | average, which is a more accurate value to characterize bacterial permeability
| |
− | property.</p>
| |
− |
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/1/1a/Ustc-npn2.png"
| |
− | alt="Figure 15: NPN uptake result in average "></p>
| |
− |
| |
− | <p>The fluorescence intensity in average of <em>E. coli</em> BL21 wild type is 34521.16. On
| |
− | the contrary, the fluorescence intensity revised by bacterial concentration in <em>E.
| |
− | coli</em> BL21 with T7-OprF (<a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>)
| |
− | and <em>E. coli</em> BL21 with T7-SCVE, (<a
| |
− | href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>) are
| |
− | respectively 85282.60 and 62195.8, which are relatively 2.5 folds and approximately 2
| |
− | folds to <em>E. coli</em> wild type, that is a significantly improvement compared to
| |
− | wild type.</p>
| |
− |
| |
− | <p>Consequently, through NPN uptake assay, we are able to conclude that the small molecule
| |
− | uptake capability of bacteria improved after our genetical modification. Thus <em>E.
| |
− | coli</em> BL21 with T7-OprF (<a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>)
| |
− | and <em>E. coli</em> BL21 with T7-SCVE, (<a
| |
− | href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>) are both able
| |
− | to use as the candidate bacteria strains for NDM measurement.</p>
| |
− |
| |
− | <h4 id="bibliography" class="scrollspy">Bibliography</h4>
| |
− | <ol>
| |
− | <li><p><em>T. Mattila-Sandholm et al.</em> Fluorometric assessment of Gram-negative
| |
− | bacterial permeabilization. <strong>Journal of Applied Microbiology</strong> 2000,
| |
− | 88, 213–219</p>
| |
− | </li>
| |
− | <li><p><em>Scott Banta et al.</em> Genetic Manipulation of Outer Membrane Permeability:
| |
− | Generating Porous Heterogeneous Catalyst Analogs in <em>Escherichia coli</em>
| |
− | dx.doi.org/10.1021/sb400202s <strong>ACS Synth. Biol.</strong> 2014, 3, 848−854</p>
| |
− | </li>
| |
− | </ol>
| |
− |
| |
− | </div>
| |
− | </div>
| |
− | <div class="col hide-on-small-only m3">
| |
− | <div class="toc-wrapper pinned">
| |
− | <ul class="section table-of-contents">
| |
− | <li>
| |
− | <a href="#Extraction_of_Permeability_Improvement_Fragements">Extraction of
| |
− | Permeability Improvement Fragement</a>
| |
− | </li>
| |
− | <div class="divider"></div>
| |
− | <li>
| |
− | <a href="#Construction_of_Permeability_Improving_Plasmid">Construction of
| |
− | Permeability Improving Plasmid</a>
| |
− | </li>
| |
− | <div class="divider"></div>
| |
− | <li>
| |
− | <a href="#Growth_Characterization">Growth Characterization</a>
| |
− | </li>
| |
− | <div class="divider"></div>
| |
− | <li>
| |
− | <a href="#ONPG_Assay">ONPG Assay</a>
| |
− |
| |
− | <div class="divider"></div>
| |
− | <li>
| |
− | <a href="#NPN_Assay">NPN Assa</a>
| |
− |
| |
− | <div class="divider"></div>
| |
− | <li>
| |
− | <a href="#bibliography">Bibliography</a>
| |
− | </li>
| |
− | </ul>
| |
− | </div>
| |
− | </div>
| |
− | </div>
| |
| </div> | | </div> |
| </div> | | </div> |
| | | |
− | <div id="Adhesion_Assay" class="row"> | + | <div id="Parts" class="row"> |
| <div class="card hoverable"> | | <div class="card hoverable"> |
| <div class="col s12"> | | <div class="col s12"> |
| <div class="card-content"> | | <div class="card-content"> |
− | <h3 id="characterization-of-optimal-conditions-on-polylysine-pll-coated-assay" class="scrollspy"> | + | <groupparts>USTC</groupparts> |
− | Characterization of Optimal Conditions on Polylysine(PLL) Coated Assay</h3>
| + | </div> |
| | | |
− | <p>To see the details on the mechanism of polylysine adhesion, please refer to <a
| + | </div> |
− | href="http://tower.im">Adhesion: Polylysine</a>.</p>
| + | |
| | | |
− | <p>To get the final PLL-coated protocol, check <a href="http://tower.im">Protocols: PLL
| |
− | Coated Assay</a> for further information.</p>
| |
− |
| |
− | <p>Our original characterization of optimal conditions on polylysine(PLL)-coated assay
| |
− | requires several factors to get, including:</p>
| |
− | <ol>
| |
− | <li>Best bacterial developmental interval along with recommended dilution conditions.
| |
− | </li>
| |
− | <li>Best PLL-Coated Concentration and Pre-treatment Time.</li>
| |
− | <li>Best PLL-Coated Time, given full consideration to the completion of bacterial
| |
− | adhesion time.
| |
− | </li>
| |
− | <li>Best Measurement Interval, which illustrates the possible time for bacterial
| |
− | response on antibiotics pressure.
| |
− | </li>
| |
− | <li>Possible Determination Interval, which refers to antibiotics response interval.</li>
| |
− | </ol>
| |
− | <p>Here we present all the optimistic conditions for users to get the effective results on
| |
− | antibiotics using our CACCI and SPRING.</p>
| |
− |
| |
− | <h3 id="Best_Bacterial_Developmental_Interval_Along_with_Recommended_Dilution_Conditions" class="scrollspy">Best Bacterial Developmental Interval Along with Recommended Dilution
| |
− | Conditions</h3>
| |
− |
| |
− | <p>According to experience on <em>E. coli</em> development based on OD detector. We
| |
− | concluded that bacteria exploding during <strong>Logarithmic Period(OD:535nm about
| |
− | 0.4~0.5)</strong> are at the most energetic moments with proper bacterial density.
| |
− | Consequently, we highly recommend user to culture bacteria at Logarithmic Period and
| |
− | then <strong>dilute bacterial solution about twenty times to fifty times.</strong></p>
| |
− |
| |
− | <h3 id="Best_PLL-Coated_Concentration_and_Pre-treatment_Time" class="scrollspy">Best PLL-Coated Concentration and Pre-treatment Time.</h3>
| |
− |
| |
− | <p>Best PLL-Coated Conditions, which include PLL-Pretreatment time, PLL-Coated concentration
| |
− | and PLL-Coated time are measured for best adhesive condictions for bacterial
| |
− | treatment.</p>
| |
− |
| |
− | <p>As for PLL-Pretreatment time, we adopt traditional pretreatment time, incubating about
| |
− | 12~16 h, for recommendation, which is originally used for neurons coated on neural
| |
− | science research. And when it comes to PLL-coated concentration, experience on neurons
| |
− | adhesion is also precious. According to previous research, PLL concentration in the
| |
− | interval of 20 ug/mL to 100 ug/mL is quite effective for bacterial adhesion.</p>
| |
− |
| |
− | <h3 id="Adhesion_Assay_with_PLL_treatment" class="scrollspy">Adhesion Assay with PLL treatment</h3>
| |
− | <p>Here we deliver the PLL treatment
| |
− | results comparing with no PLL treatment assay. During pre-experiment adhesion assay,
| |
− | PAO1, a strain of <em>Pseudomonas aeruginosa</em>, is used for adhesion effects. </p>
| |
− |
| |
− | <p>The picture below showed the amount of bacteria(PAO1) under microscope without PLL
| |
− | treatment, which is abbreviated as PLL(-). To observe capability of bacterial adhesion,
| |
− | we wash observed place with PBS:</p>
| |
− |
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/c/c3/Control_-1.png"
| |
− | alt="Figure 1: Control Assay-Without PLL treatment"></p>
| |
− |
| |
− | <p>After elution, a significant decline in the number of bacteria is recorded. As a matter
| |
− | of fact, there is no bacteria in observed field after PBS washing. </p>
| |
− |
| |
− | <p><em>To get the algorithm of bacterial counting, please refer to <a
| |
− | href="http://tower.im">Modeling: Bacterial number and movement analysis</a></em>.
| |
− | </p>
| |
− |
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/e/e9/PAO1-PLL--.png"
| |
− | alt="Figure 2: Control Assay-Without PLL treatment2"></p>
| |
− |
| |
− | <p>As for treatment with PLL in 20 ug/mL, which is abbreviated as PLL(+,20), the impressive
| |
− | adhesion effect is observed. To ensure the exact effect, bacteria observing field is
| |
− | washed twice by PBS.</p>
| |
− |
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/9/96/Adhesion_Assay.png"
| |
− | alt="Figure 3: Adhesion Assay-With PLL treatment in 20 ug/mL"></p>
| |
− |
| |
− | <p>The number of bacteria doesn't decline, and on the contrary, we see slight increase of
| |
− | bacteria number. The possible reason is constant bacterial settlement during adhesion
| |
− | assay. After twice PBS wash, the number of bacteria is relatively stable as well.
| |
− | Consequently, we concluded PLL has significant adhesive ability on bacteria.</p>
| |
− |
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/9/9e/PAO-Withoug.png"
| |
− | alt="Figure 4: Adhesion Assay-With PLL treatment in 20 ug/mL"></p>
| |
− |
| |
− | <h3 id="Best_PLL-Coated_Time" class="scrollspy">Best PLL-Coated Time</h3>
| |
− | <p>Then, we tried to figure out the cohesive effect
| |
− | of polylysine through time to get the best PLL-coated time. We gathered data just after
| |
− | PLL treatment(0s), after 1min and after 5min. And we also recorded bacteria number in
| |
− | the following 20 s. Here we provide the analysis of bacterial number variation after PLL
| |
− | treatment.We respectively use PAO1, a kind of <em>Pseudomonas aeruginosa</em> and HCB1,
| |
− | a kind of <em>E. coli</em> to handle the assay. PAO1 contains self-adhesive ability and
| |
− | HCB1 has strong mobile ability. Using these genetically natural bacteria, we would
| |
− | conclude with the effect of polylysine treatment.</p>
| |
− |
| |
− | <p><em>As for PAO1</em>, Without polylysine coated, bacteria have strong swimming ability.
| |
− | Because PAO1 is a kind of self-adhesive bacteria for experiment, thus we could see
| |
− | adhesive bacteria increasing during assay.</p>
| |
− |
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/5/5c/Ustc-2015-pll90.png"
| |
− | alt="Figure 5: no antibiotics bacterial number variation through time without PLL">
| |
− | </p>
| |
− |
| |
− | <p>With 20 ug/mL polylysine treatement, bacterial adhesive effect due to strong
| |
− | electrostatic adhesion becomes stronger, and we, as well are able to observe the number
| |
− | of adherent bacteria gradually rising until reaching equilibrium. </p>
| |
− |
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/e/ee/Developemnt.png"
| |
− | alt="Figure 6: no antibiotics bacterial number variation through time with PLL"></p>
| |
− |
| |
− | <p>After mathematical simulation we found that the adhesion rate of PAO1 by polylysine after
| |
− | treatment of PLL is increased, while for E. coli, the adhesion quantity rise, not
| |
− | fall.</p>
| |
− |
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/0/0d/USTC-PAO1.png" alt="图片名称"></p>
| |
− |
| |
− | <p><em>As for HCB1</em>, a kind of <em>E. coli</em> and the number of bacteria inside
| |
− | observed field is stable and relatively declined because of its lack of capability of
| |
− | adhesion.<br><img src="https://static.igem.org/mediawiki/2015/6/6f/Development22.png"
| |
− | alt="图片名称"></p>
| |
− |
| |
− | <p>After treated with 20ug/mL polylysine, adhesive bacteria number obviously
| |
− | increased:<br><img src="https://static.igem.org/mediawiki/2015/9/96/4141414.png" alt="图片名称">
| |
− | </p>
| |
− |
| |
− | <p>Modeling on adhesion process strictly demonstrated the adhesion assay fits Langmuir
| |
− | absorption isoform.</p>
| |
− |
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/a/a9/Ustc-bacterial_HCB1.png"
| |
− | alt="图片名称"><br><img src="https://static.igem.org/mediawiki/2015/d/d8/20150901022.png"
| |
− | alt="图片名称"><br>Simulation result:<br><img
| |
− | src="https://static.igem.org/mediawiki/2015/8/86/20150903028.png" alt=""><br>Constants
| |
− | value and details:<br><img src="https://static.igem.org/mediawiki/2015/6/66/20150903029.png"
| |
− | alt=""></p>
| |
− |
| |
− | <p>To know more about our modeling, please check <a href="http://tower.im">Modeling:
| |
− | Adhesion Assay Modeling</a></p>
| |
− |
| |
− | <p>This significant reverse of bacterial adhesion tendency stronly proved that polylysine
| |
− | has effective adhesive ability for SPRING.</p>
| |
− |
| |
− | <p>After we confirmed the feasibility of PLL treatment for bacterial adhesion, then the
| |
− | treating time should be taken into consideration because only in the case of stable
| |
− | adhesion is effective for SPRING to gather effective and stable data.</p>
| |
− |
| |
− | <p>As the plot illustrates, bacteria number is growing at the beginning of PLL treatment
| |
− | because of opening strong electrostatic adsorption derived from PLL. And after 1min, the
| |
− | number of bacteria become stable, and there is nearly no difference on bacteria number
| |
− | comparing after 1 min treatment to after 5 min treatment.</p>
| |
− |
| |
− | <p>Consequently, we recommended that PLL treatment after 5 min would be a promising set for
| |
− | SPRING to output stable data.</p>
| |
− |
| |
− | <h3 id="Best_Measurement_Interval" class="scrollspy">Best Measurement Interval</h3>
| |
− |
| |
− | <p>According to chemotaxis, bacteria responsing to surrounding pressure or beneficits will
| |
− | be presented as change of bacteria movement. Here we analyzed bacterial movement data
| |
− | treating with different antibiotics concentration, specificly, chloromycetin
| |
− | concentration. And we gathered bacterial movement data just after antibiotics
| |
− | treatment(0s), after 1 min treatment and after 5 min treatment, and we again recorded
| |
− | all pictures in the following 18 s to get the dynamic data. Then, we concluded from the
| |
− | percentage of bacterial movement as an important data to intercept the effect of
| |
− | bacterial response on antibiotics.</p>
| |
− |
| |
− | <p>The measurement assay is illustrated as following:</p>
| |
− |
| |
− | <p><em>PLL(-)</em></p>
| |
− | <table>
| |
− | <thead>
| |
− | <tr>
| |
− | <th>Antibiotics Concentration(ug/mL)</th>
| |
− | <th>Incubation Time(s)</th>
| |
− | </tr>
| |
− | </thead>
| |
− | <tbody>
| |
− | <tr>
| |
− | <td>0</td>
| |
− | <td>0</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>0</td>
| |
− | <td>60</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>0</td>
| |
− | <td>120</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>0</td>
| |
− | <td>180</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>0</td>
| |
− | <td>240</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>0</td>
| |
− | <td>300</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>1</td>
| |
− | <td>0</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>1</td>
| |
− | <td>60</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>1</td>
| |
− | <td>300</td>
| |
− | </tr>
| |
− | </tbody>
| |
− | </table>
| |
− | <p><em>PLL(+,20)</em></p>
| |
− | <table>
| |
− | <thead>
| |
− | <tr>
| |
− | <th>Antibiotics Concentration(ug/mL)</th>
| |
− | <th>Incubation Time(s)</th>
| |
− | </tr>
| |
− | </thead>
| |
− | <tbody>
| |
− | <tr>
| |
− | <td>0</td>
| |
− | <td>0</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>0</td>
| |
− | <td>60</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>0</td>
| |
− | <td>120</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>0</td>
| |
− | <td>180</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>0</td>
| |
− | <td>240</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>0</td>
| |
− | <td>300</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>0.1</td>
| |
− | <td>0</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>0.1</td>
| |
− | <td>60</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>0.1</td>
| |
− | <td>300</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>0.5</td>
| |
− | <td>0</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>0.5</td>
| |
− | <td>60</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>0.5</td>
| |
− | <td>300</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>1</td>
| |
− | <td>0</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>1</td>
| |
− | <td>60</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>1</td>
| |
− | <td>300</td>
| |
− | </tr>
| |
− | </tbody>
| |
− | </table>
| |
− | <p>When bacteria are treated with PLL, the movement of bacteria is been limited for strong
| |
− | adhesion, after 5 min treatment, the proportion of movement decreased through
| |
− | time.<br><img src="https://static.igem.org/mediawiki/2015/9/9b/-ustc-nosd.png" alt="图片名称"></p>
| |
− |
| |
− | <p>However, when bacteria are not treated with PLL, incubated in 1ug/mL chloromycetin
| |
− | solution, we got the movement percentage of bacteria as following:</p>
| |
− |
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/c/ca/Results-0.png" alt="图片名称"></p>
| |
− |
| |
− | <p>The amount of moving bacteria is relatively increasing after 5 min antibiotics treatment.
| |
− | Bacause of no PLL treatment, we could see the relative increase of bacteria movement
| |
− | percentage.</p>
| |
− |
| |
− | <p>As for bacteria treated with 20 ug/mL PLL, different pattern is observed:</p>
| |
− |
| |
− | <p>This is the bacterial movement percentage variation with time, treated with 0.1 ug/mL
| |
− | chloromycetin solution. Antibiotics pressure is not that strong, consequently, the
| |
− | movement of bacteria is not ignited significantly.</p>
| |
− |
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/b/b0/Results-1.png" alt="图片名称"></p>
| |
− |
| |
− | <p>When it comes to 0.5 ug/mL chloromycetin solution, the proportion of the moving bacteria
| |
− | is increased. And as a matter of fact, the increase is quite corresponding through time,
| |
− | reflecting bacteria impressive response on antibiotics substance.<br><img
| |
− | src="https://static.igem.org/mediawiki/2015/3/32/Ustc-2015-results-2.png" alt="图片名称">
| |
− | </p>
| |
− |
| |
− | <p>What if the concentration of chloromycetin solution up to 1 ug/mL?<br><img
| |
− | src="https://static.igem.org/mediawiki/2015/0/0a/Ustc-2015-results-4.png" alt="图片名称"><br>A
| |
− | quite linear increase of the proportion of the moving bacteria with time is observed.
| |
− | </p>
| |
− |
| |
− | <p>Consequently, according to our experiment, genetically naturally bacteria could respond
| |
− | to chloromycetin solution from 0.1 ug/mL to more than 1ug/mL, which is quite promising
| |
− | after genetic modification.</p>
| |
− |
| |
− | <p><img src="https://static.igem.org/mediawiki/2015/c/c3/Finalresult.png" alt="图片名称"></p>
| |
− | </div>
| |
− | </div>
| |
− | <div class="col hide-on-small-only m3">
| |
− | <div class="toc-wrapper pinned">
| |
− | <ul class="section table-of-contents">
| |
− | <li>
| |
− | <a href="#characterization-of-optimal-conditions-on-polylysine-pll-coated-assay">Characterization of Optimal Conditions on Polylysine(PLL) Coated Assay</a>
| |
− | </li>
| |
− | <div class="divider"></div>
| |
− | <li>
| |
− | <a href="#Best_Bacterial_Developmental_Interval_Along_with_Recommended_Dilution_Conditions">Best Bacterial Developmental Interval Along with Recommended Dilution Conditions</a>
| |
− | </li>
| |
− | <div class="divider"></div>
| |
− | <li>
| |
− | <a href="#Best_PLL-Coated_Concentration_and_Pre-treatment_Time">Best PLL-Coated Concentration and Pre-treatment Time.</a>
| |
− | </li>
| |
− | <div class="divider"></div>
| |
− | <li>
| |
− | <a href="#Adhesion_Assay_with_PLL_treatment">Adhesion Assay with PLL treatment</a>
| |
− | </li>
| |
− | <div class="divider"></div>
| |
− | <li>
| |
− | <a href="#Best_PLL-Coated_Time">Best PLL-Coated Time</a>
| |
− | </li>
| |
− | <div class="divider"></div>
| |
− | <li>
| |
− | <a href="#Best_Measurement_Interval">Best Measurement Interval</a>
| |
− | </li>
| |
− | </ul>
| |
− | </div>
| |
− | </div>
| |
− | </div>
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− | <div id="Parts" class="row">
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− | <div class="card hoverable">
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− | <div class="col s12 m9">
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− | <div class="card-content">
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− | </html>
| |
− | <groupparts>iGEM015 Example</groupparts>
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− | <html>
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− | </div>
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− | </div>
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− | </div>
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− | </div>
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− | </div>
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| </div> | | </div> |