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| <div class="info"> | | <div class="info"> |
| <!--Section title --> | | <!--Section title --> |
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| </div> | | </div> |
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| + | <!--- Project --------------------------------------------------------------------> |
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− |
| + | <div class="row"> |
| + | |
| + | <!-- Third width section with the id 'motivation' ---> |
| + | <div class="section third" id="motivation"> |
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| + | <!--Image for a third width section, recommended size: 400 x 200 px --> |
| + | <img src="https://2020.igem.org/File:T--XMU-China--Poster_03.jpg"> |
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| + | <!--Info: Place the title and text for this section, it will appear when the user clicks on it--> |
| + | <div class="info"> |
| + | |
| + | <!--Section title --> |
| + | <div class="title"> Motivation </div> |
| + | |
| + | <!--Section text---> |
| + | <div class="text"> |
| + | We identified 3 areas of synthetic biology that we wanted to improve for the iGEM community: Building, Characterizing, and Sharing. |
| + | <br><br> |
| + | |
| + | |
| + | <!--Quarter width subsection ----> |
| + | <div class="subsection quarter"> |
| + | <img src="https://static.igem.org/mediawiki/2020/a/a7/BU12_green_gear.png" align="top"> |
| + | </div> |
| + | |
| + | |
| + | <!-- Three quarters width subsection ----> |
| + | <div class="subsection three_quarters"> |
| + | <br> |
| + | <b>BUILDING:</b> BioBricks assembly requires multiple cycles of ligations and digestions, making it very time consuming when constructing large circuits |
| + | </div> |
| + | |
| + | |
| + | <!--Place a clear to make sure the next part starts on a new line ----> |
| + | <div class="clear"></div> |
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| + | <!--Quarter width subsection ----> |
| + | <div class="subsection quarter"> |
| + | <img src="https://static.igem.org/mediawiki/2020/e/ee/BU12_yellow_gear.png" align="top"> |
| + | </div> |
| + | |
| + | <!-- Three quarters width subsection ----> |
| + | <div class="subsection three_quarters"> |
| + | <br> |
| + | <b>CHARACTERIZING:</b> Characterization methods for circuits containing fluorescent proteins vary, making data comparison challenging |
| + | </div> |
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| + | <!--Place a clear to make sure the next part starts on a new line ----> |
| + | <div class="clear"></div> |
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| + | <!--Quarter width subsection ----> |
| + | <div class="subsection quarter"> |
| + | <img src="https://static.igem.org/mediawiki/2020/0/04/BU12_blue_gear.png" align="top"> |
| + | </div> |
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| + | <!-- Three quarters width subsection ----> |
| + | <div class="subsection three_quarters"> |
| + | <br> |
| + | <b>SHARING:</b> Parts pages on the Registry often lack a standard format in which characterization information is displayed |
| + | </div> |
| + | |
| + | |
| + | </div> |
| + | </div> |
| + | </div> |
| + | |
| + | |
| + | |
| + | <!-- Third width section with the id 'building_moclo' ---> |
| + | <div class="section third" id="building_moclo"> |
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| + | <!--Image for a third width section, recommended size: 400 x 200 px --> |
| + | <img src="https://2020.igem.org/File:T--XMU-China--Poster_04.jpg"> |
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| + | <!--Info: Place the title and text for this section, it will appear when the user clicks on it--> |
| + | <div class="info"> |
| + | |
| + | <!--Section title --> |
| + | <div class="title"> Building: MoClo Assembly Method </div> |
| + | |
| + | <!--Section text---> |
| + | <div class="text"> |
| + | Modular Cloning, or MoClo, is a relatively new assembly method based on Golden Gate introduced in 2011 by Ernst Weber et al., which uses Type IIS restriction enzymes (we used BpiI and BsaI) to generate 4bp overhangs. These 4bp overhangs are called fusion sites, which must match between parts in order to ligate them together. |
| + | <br><br> |
| + | This allows the user to ligate up to six DNA parts together in a one-pot reaction, cutting down the time it takes to build large circuits dramatically (shown below). |
| + | <br><br> |
| + | |
| + | <!--Half width subsection ----> |
| + | <div class="subsection half"> |
| + | <img src="https://static.igem.org/mediawiki/2020/f/f1/BioBricks_Assembly_9day_BU12.png" align="top"> |
| + | </div> |
| + | |
| + | <!--Half width subsection ----> |
| + | <div class="subsection half"> |
| + | <img src="https://static.igem.org/mediawiki/2020/c/c6/MoClo_Assembly_3day_BU12.png" align="top"> |
| + | </div> |
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| + | |
| + | There are three Levels of MoClo Parts (shown below): |
| + | <br><br> |
| + | <b>Level 0:</b> Basic part (ex: promoter, RBS, CDS, etc.)<br> |
| + | <b>Level 1:</b> Transcriptional unit (up to 6 Level 0 Parts)<br> |
| + | <b>Level 2:</b> Composite of up to 6 Level 1 parts |
| + | <br><br> |
| + | |
| + | <img src="https://static.igem.org/mediawiki/2020/0/0b/MoClo_Assembly_details_BU12.png"> |
| + | <br><br> |
| + | Reference: Weber et al., <i>PLoS One</i> 2011 |
| + | </div> |
| + | </div> |
| + | </div> |
| + | |
| + | |
| + | |
| + | <!-- Third width section with the id 'building_library' ---> |
| + | <div class="section third" id="building_library"> |
| + | |
| + | <!--Image for a third width section, recommended size: 400 x 200 px --> |
| + | <img src="https://2020.igem.org/File:T--XMU-China--Poster_05.jpg"> |
| + | |
| + | |
| + | <div class="info"> |
| + | |
| + | <!--Section title --> |
| + | <div class="title">Building: MoClo Library </div> |
| + | |
| + | <!--Section text---> |
| + | <div class="text"> |
| + | <b>We converted 31 BioBricks and 4 new parts into Level 0 MoClo parts to create a library of MoClo parts.</b> |
| + | <br><br> |
| + | |
| + | <!--Third width subsection ----> |
| + | <div class="subsection third"> |
| + | For BioBrick parts >100bp, we used a standard PCR methodology. |
| + | </div> |
| + | |
| + | <!--Two thirds width subsection ----> |
| + | <div class="subsection two_thirds"> |
| + | <img src="https://static.igem.org/mediawiki/2020/e/e7/BU12_Primer_design.png"> |
| + | </div> |
| + | |
| + | <!--Place a clear to make sure the next part starts on a new line ----> |
| + | <div class="clear"></div> |
| + | |
| + | <!--Third width subsection ----> |
| + | <div class="subsection third"> |
| + | We used a ligation PCR methodology for sequences < 100bp in length (J. Lee et al., Biotechniques. 2004). |
| + | </div> |
| + | |
| + | <!--Two thirds width subsection ----> |
| + | <div class="subsection two_thirds"> |
| + | <img src="https://static.igem.org/mediawiki/2020/9/93/BU12_ligation_pcr.png"> |
| + | </div> |
| + | |
| + | <!--Place a clear to make sure the next part starts on a new line ----> |
| + | <div class="clear"></div> |
| + | |
| + | <br> |
| + | |
| + | <!--Three quarters width subsection ----> |
| + | <div class="subsection three_quarters"> |
| + | <img src="https://static.igem.org/mediawiki/2020/8/8a/BU12_Table_1_parts_updated.png"> |
| + | </div> |
| + | |
| + | All parts listed in Table 1 were confirmed with sequencing |
| + | <br><br> |
| + | All of our parts and primers are stored in a Clotho database (Xia et al., Methods in Enzymology 2011). |
| + | <br><br><br><br><br> |
| + | |
| + | <b>New Parts</b> |
| + | <br> |
| + | We have cloned a new copper sensitive σ<sup>54</sup>-regulatory system: pMmoR is induced by mmoR, which is repressed by copper (J. Scanlan et al., 2009). |
| + | <br><br> |
| + | Two new fluorescent proteins, EBFP2 and iRFP, have also been cloned. |
| + | |
| + | </div> |
| + | </div> |
| + | </div> |
| + | |
| + | </div> |
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