Modular Cloning (MoClo) Guide

Overview

Synthetic biologists have developed a strategy of modular cloning, called MoClo, to efficiently assemble multiple DNA parts into functional plasmids. It is a powerful and very efficient method for creating many plasmids from different combinations of a common set of components (Weber et al., 2011). This method (based on Golden Gate plasmid assembly) exploits the ability of Type IIS restriction enzymes BsaI and BpiI/BbsI to cut outside their recognition site, leaving DNA fragments with compatible overhangs. If each DNA module is flanked with a unique overhang at the restriction cut site, multiple modules can be digested and ligated in a single step while retaining only the 4-bp fusion site in between.

The MoClo system consists of three sets of cloning vectors (Level 0, 1, and 2) which can be used in successive assembly steps. Before beginning, scientists insert fragments of DNA containing basic parts (promoters, UTRs, coding sequences, terminators, etc.) flanked by fusion sites 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 parts are directionally assembled into a Level 1 vector creating a single transcriptional unit (for example, a promoter, a 5'UTR, a signal peptide, a protein-coding gene, and a terminator, as in Figure 1). Next, up to six Level 1 modules can be assembled into a Level 2 vector, forming a functional genetic circuit. Level 2 vectors are often designed with flexibility to allow for additional iterations of assembly. Combining multiple Level 2 vectors permits the creation of even more complex constructs constrained only by the ability of E. coli to maintain the final plasmid after transformation.

MoClo can be used across a variety of applications in various organisms—like building synthetic genetic circuits, metabolic engineering, assembling gene-editing tools, and more. Explore the following list of MoClo plasmid kits that have been deposited with Addgene.

• Figure 1: Overview of the MoClo Toolkit components and assembly.
• Image from Weber et al., 2011.

MoClo Plasmid Kits Available from Addgene

Plant Expression

• MoClo Toolkit (deposited by Sylvestre Marillonnet) - 95 plasmids for cloning and assembling standardized parts to create eukaryotic multigene constructs.

• MoClo Plant Parts Kit (deposited by Nicola Patron) - 95 standardized parts specific for plant synthetic biology.

• MoClo Plant Parts II and Infrastructure Kit (deposited by Johannes Stuttmann) - 95 additional plasmids for hierarchical assembly of plant multigene constructs.

• MoClo Plant Parts III: Transformation & Genome Engineering Kit (deposited by Daniel Voytas) - 93 additional plasmids including regulatory elements, coding sequences, CRISPR parts

• GreenGate Cloning System (deposited by Jan Lohmann) - Plasmids to create plant expression vectors containing several cassettes and generate multi-construct transgenic plants.

• GreenGate 2.0 Toolkit (deposited by Alexis Maizel) - 68 plasmids to extend the GreenGate Cloning System (Lohmann) with additional Level 0 modules and components to create plasmids with multiple transcriptional units.

• GoldenBraid 2.0 Kit (deposited by Diego Orzaez) - 94 plasmids using the GoldenBraid framework for plant synthetic biology and multigene engineering, including CRISPR/Cas9 components.

• MoChlo: Modular Cloning Chloroplast Toolbox (deposited by Scott Lenaghan) - 122 plasmids with chloroplast-specific genetic modules, including destination vectors specific for tobacco (N. tabacum) or potato (S. tuberosum).

• Joint Modular Cloning (JMC) Toolkit (deposited by Daniel Voytas) - 76 plasmids for assembling plant transformation vectors for overexpression, gene editing, functional genomics, and more complex metabolic engineering and synthetic biology, compatible with PhytoBrick parts.

Yeast and Fungal Expression

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

• Multiplex Yeast Toolkit (MYT) (deposited by Tom Ellis) - 96 plasmids to extend the MoClo-YTK (Dueber) for genetic engineering of S. cerevisiae, enabling larger multi-gene constructs, multiplexed CRISPR editing, and more.

• Fungal Toolkit for Modular Cloning (FTK) (deposited by Arnold Driessen) - 96 plasmids (including CRISPR/Cas9 components) for expression or gene editing in filamentous fungi such as Penicillium or Aspergillus species.

• MoClo Yeast Secretion and Display (YSD) Toolkit (deposited by Paul Young) - 34 plasmids that can be used in conjunction with the MoClo Yeast Toolkit (YTK) to optimize secretion and cell surface display of proteins of interest.

• Yeast Secrete and Detect (deposited by Sylvestre Marillonnet) - Plasmids to generate transcription units for protein secretion in yeasts S. cerevisiae and P. pastoris.

• MoClo Pichia Toolkit (deposited by Volker Sieber) - Plasmids with control elements and fluorescent reporters to be used in combination with the MoClo-YTK toolkit for protein expression and secretion in P. pastoris.

• GoldenPiCS Kit (deposited by Brigitte Gasser, Diethard Mattanovich, and Michael Sauer) - 62 plasmids for advanced strain engineering in P. pastoris, including overexpression plasmids with up to eight transcription units.

• Yarrowia lipolytica Golden Gate tool kit (deposited by Tristan Rossignol) - 64 plasmids to assemble and integrate up to three transcription units in the oleaginous yeast Y. lipolytica.

• Yeast GPCR-sensor Toolkit (deposited by Tom Ellis) - Plasmids for constructing highly-tunable GPCR-based biosensors in yeast (requires MoClo-YTK parts for use).

• Yeast GoldenBraid Cloning System and Toolkit (deposited by Elena Caro Bernat) - Plasmids for generating transcriptional units using the GoldenBraid system, for integration in S. cerevisiae.

• Easy-MISE Toolkit (deposited by Paola Branduardi) - Easy-Modular Integrative fuSion-ready Expression toolkit for improved heterologous protein production in S. cerevisiae.

Bacterial Expression

• EcoFlex MoClo Toolkit (deposited by Paul Freemont) - 78 plasmids including 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) - A collection of 93 modular plasmids and enhanced MoClo protocols for rapid one-pot, multipart assembly, combinatorial design, and expression tuning in E. coli.

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

• openCIDAR MoClo Toolkit (deposited by Eric Young) - Broad host range vectors compatible with the CIDAR MoClo cloning standard, including a combinatorial library of constructs expressing GFP at 18 levels for rapid characterization in new organisms.

• Mobius Assembly Vector Toolkit (deposited by Naomi Nakayama) - A toolkit with both high cloning capacity and vector simplicity, compatible with other MoClo, GoldenBraid, and PhytoBricks standards.

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

• Cultivarium POSSUM Toolkit (deposited by Nili Ostrov, Charlie Gilbert, and Cultivarium Tools) - 189 plasmids, including origins of replication and selection markers for a variety of bacterial and fungal species for engineering non-model microbes.

• AspFlex Kit (deposited by Veronica Godoy-Carter) - Plasmids for protein expression and CRISPRi experiments in Acinetobacter spp. and Acinetobacter baumannii.

• Komagataeibacter Tool Kit (KTK) (deposited by Tom Ellis) - DNA parts to create multigene constructs expressed in the cellulose-producing Komagataeibacter species.

• Ralstonia-GG-Kit (deposited by Thomas Lahaye) - Plasmids to generate gene knockout and complementation constructs for use in Ralstonia solanacearum.

• Ubigate Collection (deposited by Marco Trujillo) - Plasmids for reconstituting the ubiquitination cascades of different organisms in E. coli.

• MetClo DNA Assembly Kit (deposited by Christopher A. O'Callaghan) - Toolkit for assembling large DNA fragments based on methylation-switching.

Mammalian Expression

• The Mammalian Toolkit (deposited by Hana El-Samad) - 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.

• EMMA: Extensible Mammalian Modular Assembly Toolkit (deposited by Yizhi Cai) - 95 plasmids to generate mammalian expression vectors for transient or stable transfections.

• PUF Assembly Kit (deposited by Huimin Zhao) - Plasmids to assemble custom-made RNA-binding proteins based on the Pumilio and FBF (PUF) homology domain, for mammalian or E. coli expression.

Genome Engineering

• Multiplex CRISPR/Cas9 Assembly Systems (deposited by Takashi Yamamoto) - Plasmids for construction of all-in-one CRISPR/Cas9 vectors expressing multiple gRNAs (2–7) with a Cas9 nuclease, nickase, dCas9, or dCas9-FokI fusion for use in human or mouse systems.

• zCas9i cloning kit: MoClo-compatible CRISPR/Cas9 cloning kit with intronized Cas9 (deposited by Sylvestre Marillonnet and Johannes Stuttmann) - 73 plasmids for assembly of CRISPR/Cas9 constructs for use in plants. The constructs are made with zCas9i, a high efficiency intron-optimized SpCas9-coding gene.

• MoClo CRISPR/Cas Toolkit for Plants (deposited by Vladimir Nekrasov) - 95 plasmids including CRISPR/Cas nucleases, base editors, gRNA backbones, and promoters for genome editing in monocots and dicots.

• pDGE Dicot Genome Editing Kit (deposited by Johannes Stuttmann) - Plasmids for assembly of constructs for CRISPR genome editing in dicotyledonous plants.

• CRISPi Kit (deposited by Brigitte Gasser) - Plasmids to perform CRISPR/Cas9 mediated genome editing in the yeast P. pastoris.

• NT-CRISPR Plasmid Collection (deposited by Anke Becker) - Plasmids to perform genome engineering in Vibrio natriegens.

• TALE Toolbox (deposited by Feng Zhang) - Plasmids for building TALE nucleases (TALENs) and transcription factors (TALE-TFs) for genome editing and transcription modulation applications.

• Golden Gate TALEN and TAL Effector Kit 2.0 (deposited by Daniel Voytas and Adam Bogdanove) - Plasmid toolkit to efficiently assemble TALEN constructs with custom repeat arrays, containing anywhere between 12 and 31 repeats.

• Golden Gate TALEN Accessory Pack (deposited by Takashi Yamamoto) - Nine pFUS array vectors and destination vectors designed for use with the Golden Gate TALEN and TAL Effector Kit (Voytas and Bogdanove).

• Platinum Gate TALEN Kit (deposited by Takashi Yamamoto) - Plasmids for construction of highly-active Platinum TALENs for use in a wide variety of target organisms.

• TALEN Kit (deposited by Stephen Ekker) - 256 plasmids encoding all possible combinations of pFUS_B4 clones for Golden Gate assembly of 15-RVD TALENs.

• FusX TALEN Assembly System Kit (deposited by Stephen Ekker) - 336 plasmids of pre-assembled pFusX clones allowing for Golden Gate assembly of 15-16 RVD TALEN inserts in one cloning step rather than two.

Other

• Golden GATEway Cloning Kit (deposited by Joachim Wittbrodt) - A toolkit that combines Golden Gate and Multisite Gateway cloning. This system was specifically designed for generating zebrafish transgenesis constructs, but is also suitable for creating fusion proteins for use in many different model organisms.

• Diatom uLoop Assembly Kit (deposited by Christopher Dupont) - 95 plasmids including backbones, fluorescent proteins, protein tags, and signal peptides, for use in a range of organisms including diatoms, yeast, plants and bacteria.

References and More Resources

• Guide to DNA Assembly Techniques by Nicola Patron at The Sainsbury Laboratory

• Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A modular cloning system for standardized assembly of multigene constructs. PLoS One. 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765. PMID: 21364738 .

• Engler C, Youles M, Gruetzner R, Ehnert TM, Werner S, Jones JD, Patron NJ, Marillonnet S. A golden gate modular cloning toolbox for plants. ACS Synth Biol. 2014 Nov 21;3(11):839-43. doi: 10.1021/sb4001504. PMID: 24933124 .