Team:BostonU/MoCloChara

From 2013.igem.org

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<h9>MoClo Parts</h9>
<h9>MoClo Parts</h9>
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<p dir="ltr">The MoClo system has three levels of assembly.
<p dir="ltr">The MoClo system has three levels of assembly.
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<div id="Level0">
 
<p dir="ltr">Level 0: Basic DNA Parts (ex: promoter, gene, etc.) are PCR amplified and cloned into MoClo destination vectors to form Level 0 Parts. The DNA parts within these Level 0 Parts are flanked by BsaI sites and two different 4pb-fusion sites.</p><br>
<p dir="ltr">Level 0: Basic DNA Parts (ex: promoter, gene, etc.) are PCR amplified and cloned into MoClo destination vectors to form Level 0 Parts. The DNA parts within these Level 0 Parts are flanked by BsaI sites and two different 4pb-fusion sites.</p><br>
<p><center><img src="https://static.igem.org/mediawiki/2012/d/d6/MoClo_details.png" width="575px"></a></p>
<p><center><img src="https://static.igem.org/mediawiki/2012/d/d6/MoClo_details.png" width="575px"></a></p>
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<h7><p dir="ltr">Figure 1 shows how the Type IIS enzyme BpiI (yellow blocks in Figures 1 and 2) is used to generate Level 0 Parts using MoClo. Type IIS restriction enzymes recognize their site and then cut outside of it. BpiI (and BsaI shown as purple blocks in Figures 1 and 2) both leave 4bp overhangs behind after cutting (Figure 1). We refer to the 4bp overhangs as fusion sites and based our designs on the original fusion sites described by <a href="http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0016765#pone.0016765-Engler1"<>Weber et al., 2011</a>. The orientation of BpiI and BsaI for the parts being ligated into the destination vectors face inwards while the BpiI and BsaI orientation is the opposite in the destination vector. This direction is shown in Figure 1 as the black arrows above the yellow BpiI blocks. This orientation guarantees that the BpiI sites are removed in the ligated Level 0 Part(Figure 1). The BsaI sites are likewise removed from Level 1 Parts (Figure 2).
<h7><p dir="ltr">Figure 1 shows how the Type IIS enzyme BpiI (yellow blocks in Figures 1 and 2) is used to generate Level 0 Parts using MoClo. Type IIS restriction enzymes recognize their site and then cut outside of it. BpiI (and BsaI shown as purple blocks in Figures 1 and 2) both leave 4bp overhangs behind after cutting (Figure 1). We refer to the 4bp overhangs as fusion sites and based our designs on the original fusion sites described by <a href="http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0016765#pone.0016765-Engler1"<>Weber et al., 2011</a>. The orientation of BpiI and BsaI for the parts being ligated into the destination vectors face inwards while the BpiI and BsaI orientation is the opposite in the destination vector. This direction is shown in Figure 1 as the black arrows above the yellow BpiI blocks. This orientation guarantees that the BpiI sites are removed in the ligated Level 0 Part(Figure 1). The BsaI sites are likewise removed from Level 1 Parts (Figure 2).
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<div id="Level1">
 
<p dir="ltr">Level 1: Up to six Level 0 Parts are ligated together to form Level 1 Parts. In our lab, Level 1 Parts most often result in complete transcriptional units (ex: promoter-RBS-gene-terminator). Level 1 Parts are flanked by BpiI sites (shown as yellow blocks in Figure 2) and two different 4pb-fusion sites.</p></ul>
<p dir="ltr">Level 1: Up to six Level 0 Parts are ligated together to form Level 1 Parts. In our lab, Level 1 Parts most often result in complete transcriptional units (ex: promoter-RBS-gene-terminator). Level 1 Parts are flanked by BpiI sites (shown as yellow blocks in Figure 2) and two different 4pb-fusion sites.</p></ul>
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<h3><b>Figure 2:</b> Generation of Level 0 and Level 1 MoClo Parts. PCR products are cloned into Level 0 destination vectors using the one-pot MoClo reaction with BpiI as the restriction enzyme. Level 1 composite parts are then made by mixing all four Level 0 parts together with a Level 1 destination vector and carrying out the MoClo reaction with BsaI as the restriction enzyme. Along with blue/white screening, chloramphenicol (CamR) is used to select Level 0 and kanamycin (KanR) is used to select Level 1 colonies.</h3>
<h3><b>Figure 2:</b> Generation of Level 0 and Level 1 MoClo Parts. PCR products are cloned into Level 0 destination vectors using the one-pot MoClo reaction with BpiI as the restriction enzyme. Level 1 composite parts are then made by mixing all four Level 0 parts together with a Level 1 destination vector and carrying out the MoClo reaction with BsaI as the restriction enzyme. Along with blue/white screening, chloramphenicol (CamR) is used to select Level 0 and kanamycin (KanR) is used to select Level 1 colonies.</h3>
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<h9>MoClo Simplified</h9>
<h9>MoClo Simplified</h9>
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<div id="MCContrib">
 
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<h9>Our MoClo Contribution to iGEM</h9>
<h9>Our MoClo Contribution to iGEM</h9>

Revision as of 15:38, 6 June 2013

BostonU iGEM Team: Welcome