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Modular Cloning (MoClo) Guide


Synthetic biologists have developed a modular cloning strategy, MoClo, which uses the Type IIS restriction enzymes BsaI and BpiI/BbsI to efficiently assemble up to six DNA fragments at a time. This method (based on the Golden Gate technology) exploits the ability of Type IIS enzymes to cut outside their recognition site, and permits DNA fragments with compatible overhangs to be efficiently assembled. Scientists can engineer unique enzyme recognition sites that flank a DNA module in an inverse orientation, so that multiple DNA components can directionally assemble in a single reaction, while retaining only a defined 4bp fusion site in between.

The MoClo system is comprised of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly steps. Before beginning, scientists can insert fragments of DNA containing basic parts (promoters, UTRs, coding sequences, terminators, etc) into individual Level 0 plasmids, or choose from a growing number of libraries containing pre-constructed standardized modules. In the first assembly step, compatible Level 0 vectors are directionally assembled into a Level 1 vector creating a single transcriptional unit (Ex: a promoter, 5’UTR, coding region, and terminator). Next, up to six Level 1 modules can be similarly assembled into a Level 2 vector, thus forming a functional genetic circuit. Flexibility has been built into the Level 2 vectors to allow for additional iterations of Level 1 assembly if necessary. Combining multiple Level 2 vectors in the final assembly step permits the creation of more complex constructs constrained only by the ability of E. coli to maintain the final plasmid after transformation.

MoClo Plasmid Kits Available from Addgene

  • EcoFlex MoClo Toolkit (deposited by Paul Freemont's lab)
  • - The EcoFlex Toolkit is a collection of plasmids that features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins for use in E. coli for a variety of applications.

  • CIDAR MoClo Parts Kit (deposited by Douglas Densmore's lab)
  • - Collection of modular DNA parts and enhanced MoClo protocols to enable rapid one-pot, multipart assembly, combinatorial design and expression tuning in E. coli.

  • CIDAR MoClo Extension, Volume I (deposited by Richard Murray's lab)
  • - 110 plasmids designed to quickly and efficiently build a wide variety of gene expression constructs using the CIDAR-standard MoClo system.

  • MoClo-YTK (deposited by John Dueber's lab)
  • - A set of 96 standardized and characterized parts that can be used for bottom-up hierarchical assembly of single and multi-gene constructs for S. cerevisiae expression.

  • The Mammalian Toolkit (deposited by Hana El-Samad's lab)
  • - This kit includes 253 plasmids that allow for the assembly of cellular circuits using Golden Gate-based cloning methods for use in mammalian cells. These can be used with plasmids from the MoClo-YTK.

  • CyanoGate Kit (deposited by Alistair McCormick's lab)
  • - Kit containing 96 parts and acceptor vectors for generating multi-part assemblies in either integrative or self-replicating plasmid vectors for working in cyanobacteria.

  • MoClo Toolkit (deposited by Sylvestre Marillonnet's lab)
  • - Empty plasmids for cloning and assembling standardized parts to create eukaryotic multigene constructs

  • MoClo Plant Parts Kit (deposited by Nicola Patron's lab)
  • - Contains standardized parts specific for plant synthetic biology

Image from Weber et al., PLoS One. 2011 Feb 18;6(2):e16765.

Useful Links

  • DNA Assembly Guide from SYNBIO@TSL and Nicola Patron

  • A Modular Cloning System for Standardized Assembly of Multigene Constructs. Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. PLoS One. 2011. Feb 18;6(2):e16765. PubMed.

  • A Golden Gate Modular Cloning Toolbox for Plants. Engler C, Youles M, Grützner R, Ehnert T-M, Werner S, Jones JDG, Patron NJ, Marillonnet S. ACS Synth. Biol. 2014. PubMed.