Team:BostonU/MoClo

From 2014.igem.org

• TEAM
• PROJECT
  • Overview
  • Priority Encoder
  • MoClo Assembly Method
  • Low Copy Backbones
  • Tandem Promoters
  • Fusion Proteins
  • Repressor Proteins
  • Multiplexing
  • BioDesign Automation Tools
  • Future Work
• ACHIEVEMENTS
  • Data Collected
  • Parts Submitted
  • Chimera Workflow
  • Chimera Example
  • Medal Fulfillment
• NOTEBOOK
  • Training
  • Protocols
  • Backbones
  • Tandem Promoters and Repressors
  • Fusion Proteins
• CONSIDERATIONS
  • Collaborations
  • Measurement Track
  • Safety
  • Interlab Study
  • NEGEM
  • Policy and Practices
• ACKNOWLEDGEMENTS

MoClo Assembly

Modular Cloning, or MoClo, is a relatively new assembly method introduced in 2011 by Ernst Weber et al., whereby using Type IIS restriction sites, the user can ligate at least six DNA parts together into a backbone in a one-pot reaction. It is a method based on Golden Gate Assembly, where Type IIS restriction enzymes cleave outside of their recognition site to one side, allowing for removal of those restriction sites from the design. 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-base pair fusion sites, which remain between parts after ligation, forming 4-base pair scars between DNA parts in the final DNA sequence following ligation of two or more parts. MoClo Overview The MoClo system allows directional assembly of DNA parts through using BsaI and BbsI (also called BbsI), two Type IIS restriction enzymes that leave 4bp overhangs after they cut DNA. These four base pair overhangs, or fusion sites, created by either enzyme can be any 4 bases and thus fusion sites can be user-defined. The fusion sites are used to enable directional assembly of DNA Parts. By defining non-palindromic fusion sites that flank each DNA Part, we can ensure proper order of assembly by assigning the same fusion sites between the 3’ and 5’ ends of two DNA Parts that are meant to go next to one another. MoClo Destination Vectors The MoClo system we're using has three levels of assembly where basic parts (Level 0) are linked together to form transcriptional units (Level 1), which are then linked together to form functional genetic circuits (Level 2). Each MoClo level also requires Destination Vectors which act as the end carrier for the inserted DNA part(s). In our assembly, we utilize blue/white screening for each level along with different antibiotic resistance markers as a secondary selection method for screening our clones. We have converted the common BioBrick vectors pSB1C3, pSB1K3, and pSB1A2 into our MoClo Destination Vectors by inserting the alpha fragment of lacZ with flanking MoClo restriction and fusion sites into the cloning site (Fig. 1). Figure 1: Destination Vector Detail for MoClo Levels 0-2. The BsaI and BbsI (both in gray) sites are interchanged between levels. The green backbone in Level 0 denotes chloramphenicol resistance, the orange for Level 1 is kanamycin, and the blue for Level 2 is ampicillin. The alpha lacZ fragment is in the cloning site for each level and the 4 blue N’s within the gray circles denote the fusion sites, to be designed by the user. MoClo Parts As mentioned above, our 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. During the PCR reaction, the correct fusion sites and BbsI sites are added to either side of the DNA sequence. The orientation of the BbsI sites is crucial, where the 3’ BbsI site is inverted while the 5’ BbsI site is not (Fig. 3). This PCR reaction is mixed with its matching Destination Vector in a MoClo reaction. After a transformed clone has been confirmed, this plasmid is now called a Level 0 Part and has the following order of DNA sequences: BsaI site-Fusion site 1-DNA Part-Fusion site 2-BsaI inverted (Fig. 3). Notice that BbsI is removed from the final product, thus requiring the Destination Vector to contain both BbsI and BsaI flanking the fusion sites (Fig. 2). Figure 2: Construction of a Level 0 MoClo Part. This is a specific example for creating a level 0 MoClo part with the C and D fusion sites (blue and green circles, respectively) on the 5' and 3' sides, respectively, of the DNA part. PCR primers are designed as shown in the top of the figure to include 2bp (NN), then the 6bp BbsI site (in gray), 2bp (NN), then the 4-bp fusion site (in half colored circles), followed by 18-24bp of the DNA part of interest. Once amplified with high fidelity DNA polymerase, the PCR product is mixed with the Level 0 Destination Vector that has matching fusion sites in a MoClo reaction to create the final Level 0 MoClo Part shown in the final plasmid. Level 1: Up to six Level 0 Parts can be ligated together to form Level 1 Parts. In our lab, we build Level 1 Parts with four Level 0 parts to create complete transcriptional units (ex: promoter-RBS-gene-terminator). Level 1 Parts are flanked by BbsI sites and two different 4pb-fusion sites (Fig. 3). Figure 3: Construction of a Level 1 MoClo Part. Four basic DNA Parts (promoter, RBS, CDS, and terminator) 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 fifth 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 (gray text) are shown in the DNA Part vectors and Destination Vector with the direction they cut (gray arrow). The 4bp fusion sites (text in colored circles) 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. 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 (Fig. 4). Figure 4: Construction of a Level 2 MoClo Part. Four Level 1 Transcriptional Unit Parts (made up of a promoter, RBS, CDS, and terminator) are each shown in orange vectors to denote the kanamycin resistance cassette and each has two 4bp non-palindromic fusion sites flanking the DNA Part. The fifth vector is the Destination Vector for the final construct, which is blue to denote the ampicillin resistance cassette and contains lacZ for blue-white screening. The BbsI sites (gray text) are shown in the DNA Part vectors and Destination Vector with the direction they cut (gray arrow). The 4bp fusion sites (text in colored circles at the ends of the transcriptional units) shown in the five top plasmids indicate the fusion sites that will remain with either the Level 1 Transcriptional Units or the Destination Vector after the BbsI digestion. References [1] Weber et al. (2011) "A Modular Cloning System for Standardized Assembly of Multigene Constructs." PLoS ONE 6(2): e16765. doi:10.1371/journal.pone.0016765 [2] Engler et al. (2009) "Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes." PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 [3] Haddock, T., and Densmore, D. (2013) BBF RFC 94: The Modular Cloning Assembly: Standardized Assembly of Bacterial Transcriptional Units Using Type IIS Restriction Enzymes BsaI and BpiI.

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