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)
- CIDAR MoClo Parts Kit (deposited by Douglas Densmore's lab)
- MoClo-YTK (deposited by John Dueber's lab)
- MoClo Toolkit (deposited by Sylvestre Marillonnet's lab)
- MoClo Plant Parts Kit (deposited by Nicola Patron'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.
- Collection of modular DNA parts and enhanced MoClo protocols to enable rapid one-pot, multipart assembly, combinatorial design and expression tuning in E. coli.
- 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.
- Empty plasmids for cloning and assembling standardized parts to create eukaryotic multigene constructs
- Contains standardized parts specific for plant synthetic biology
Image from Weber et al., PLoS One. 2011 Feb 18;6(2):e16765.
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.