Team:BostonU/MoClo2

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<h7><p dir="ltr">A more simplified way to look at MoClo is to focus on the fusion sites. To make this easier, we labeled our 4bp fusion sites A-H.</p></ul></h7>
<h7><p dir="ltr">A more simplified way to look at MoClo is to focus on the fusion sites. To make this easier, we labeled our 4bp fusion sites A-H.</p></ul></h7>
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<h3><b>Figure 3:</b> Figure legend</h3>
<h3><b>Figure 3:</b> Figure legend</h3>

Revision as of 04:28, 12 September 2012

BostonU iGEM Team: Welcome


Modular Cloning




An Introduction to MoClo

    Modular Cloning, or MoClo, is a relatively new assembly method introduced in 2011 by Ernst Weber et al., whereby using Type IIS restriction sites allows the user to ligate up to six DNA parts together in a one-pot reaction. It is a method based on Golden Gate Assembly. Type IIS restriction enzymes cleave outside of their recognition site to one side, thus allowing for removal of those restriction sites when used properly. This helps eliminate excess base pairs, or scars, from forming between DNA Parts. However, in order to ligate together properly, MoClo utilizes a set of 4-bp fusion sites, which remain behind after ligation and thus generate 4-bp scars between DNA parts in the final DNA sequence following ligation of two or more parts.


Modules

    The MoClo system has three levels of assembly.

    Level 0: Basic DNA Parts (ex: promoter, gene, etc.) are PCR amplified and then cloned into MoClo destination vectors to form Level 0 Modules. The DNA parts within these Level 0 Modules are flanked by BsaI sites and two different 4pb-fusion sites.



    Figure 1: Generation of Level 0 Modules. The red DNA sequence indicates the BpiI restriction site, the blue DNA sequence indicates the 4-bp fusion sites, and the bracketed names indicate either GFP and LacZ gene sequences. The black arrows over the red DNA sequence indicates the BpiI direction, which impacts how the enzyme will cut the double stranded DNA sequence.


    Figure 1 shows how the Type IIS enzyme BpiI (yellow blocks in Figures 1 and 2) is used to generate Level 0 Modules 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. We refer to the 4bp overhangs as fusion sites and based our designs on the original fusion sites described by Weber et al., 2011. 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 Module (Figure 1). The BsaI sites are likewise removed from Level 1 Modules (Figure 2).

    Level 1: Up to six Level 0 parts are ligated together to form Level 1 Modules. In our lab, Level 1 Modules most often result in complete transcriptional units (ex: promoter-RBS-gene-terminator). Level 1 Modules are flanked by BsaI sites (shown as purple blocks in Figure 2) and two different 4pb-fusion sites.



    Figure 2: Figure legend



    Level 2: Up to six Level 1 Modules are ligated together to form Level 2 Modules. More complex circuits, such as an inverter or NOR gate, can be built using Level 2 Modules. Like Level 0 Modules, Level 2 Modules are flanked by BsaI sites and two different 4pb-fusion sites.


    MoClo Simplified

    A more simplified way to look at MoClo is to focus on the fusion sites. To make this easier, we labeled our 4bp fusion sites A-H.



    Figure 3: Figure legend



    Figure 4: Figure legend

    Wrap up...