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Team:BostonU/MoCloChara
From 2013.igem.org
MoClo Library Characterization
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.
MoClo Parts
The MoClo system has three levels of assembly.
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.
Figure 1: Directional Assembly of DNA Parts. Two basic DNA Parts (DNA Part 1 and 2) are each shown in green vectors to denote the chloramphenicol resistance cassette and each has two 4bp non-palindromic fusion sites flanking the DNA Part. The third vector is the Destination Vector for the final construct, which is orange to denote the kanamycin resistance cassette and contains lacZ for blue-white screening. The BsaI sites (red text) are shown in the DNA Part vectors and Destination Vector with the direction they cut (red arrow). The 4bp fusion sites (blue text) shown in the five top plasmids indicate the fusion sites that will remain with either the DNA Parts or the Destination Vector after the BsaI digestion. By allowing matching fusion sites between the 3’ end of the first part and the 5’ end of the second part and so on, this enables the proper assembly order of the DNA parts to be maintained. The Destination Vector contains the fusion site on the 5’ end of the promoter and the 3’ end of the terminator to allow the properly ordered transcriptional unit to be cloned into a vector containing a different antibiotic resistance from the vectors containing the basic DNA Parts. The DNA parts replaces the lacZ coding sequence, thus allowing for the selection of white colonies on plates containing IPTG and X-Gal. (Figure from BBF RFC 94: The Modular Cloning Assembly: Standardized Assembly of Bacterial Transcriptional Units Using Type IIS Restriction Enzymes BsaI and BpiI, Traci Haddock and Douglas Densmore, 2013).
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 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 Part(Figure 1). The BsaI sites are likewise removed from Level 1 Parts (Figure 2).
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.
Figure 2: 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.
Level 2: Up to six Level 1 Parts are ligated together to form Level 2 Parts. More complex circuits, such as an inverter or NOR gate, can be built using Level 2 Parts. Like Level 0 Parts, Level 2 Parts 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. This also allows for easier generation of images by removing the backbone and restriction site information.
Figure 3: Levels 0 and 1 MoClo Part Generation Simplified. This figure illustrates how Level 0 and Level 1 MoClo parts are generated with a focus on the 4-bp fusion sites shown in circles labeled A-H. The BpiI and BsaI sites are not shown.
Figure 4: Level 2 Part Generation Simplified. This figure illustrates how Level 2 MoClo parts are generated from Level 1 parts with a focus on the 4-bp fusion sites shown in circles labeled A-H. The BpiI and BsaI sites are not shown.
Our MoClo Contribution to iGEM
MoClo is a highly modular cloning system that we think would greatly improve the efficiency of genetic circuit generation for iGEM teams. We have designed our MoClo parts for easy, 4-way assembly of transcriptional units. In order to achieve this, our promoters are flanked by either A-B, E-B, F-B, or G-B fusion sites; our ribosomal binding sites are flanked by B-C fusion sites; our genes are flanked by C-D fusion sites; and our terminators are flanked by either D-E, D-F, D-G, or D-H fusion sites (Figure 4). By promoting this 4-way assembly, we have increased the usability of this system by keeping the number of destination vectors needed low and the PCR design stage is also simplified for the RBS and genes parts.
We have submitted a MoClo kit of parts to the Registry so future iGEM teams can take advantage of this easy, efficient system. It is also our hope that future iGEM teams will help build upon this first attempt at generating a library of standard biological parts for the MoClo system.
[1] Weber et al. "A Modular Cloning System..." PLoS One 6(2) 2011
