Plasmids 101: Modular Cloning Applications and Kits

Modular cloning is a popular DNA assembly tool used to build single- and multi-gene constructs for a variety of applications. MoClo tools can be used in bacterial and cell-free expression systems, mammalian cells, yeast, flies, and even plants! In this blog we will review common uses of MoClo and the kits Addgene offers to kickstart these applications, no matter what your model organism is.

MoClo background

MoClo is a clever way to assemble several DNA parts in a specific orientation using Type IIS restriction enzymes (Weber et al., 2011). It can be performed at multiple levels to generate anything from individual transcription units (TUs) used to express your favorite gene-of-interest, all the way to the complex multi-TU constructs that compose genetic circuits and metabolic pathways. For many MoClo systems, the individual parts are reusable, saving time and money on future assemblies. If you are new to MoClo, give our MoClo 101 blog a read and then come back here once you are up to speed on the basics of the technology.

In this blog, we will highlight some common MoClo applications and kits. Keep in mind that this technology is useful for a variety of purposes beyond what we discuss. If you can think it, MoClo might help you achieve it!

MoClo applications and specialized tools

Synthetic biology in bacteria

MoClo is commonly used to design TUs and multi-TU constructs for synthetic biology applications in bacteria or cell-free systems. The approach is particularly useful for producing drug precursors, biofuels, and natural products without the undesirable side products generated during chemical synthesis of these molecules. To make large amounts of pure product in an affordable way, researchers test many different combinations of promoters, terminators, solubility and purification tags, connectors, cDNAs, and more to find the perfect combination.

The EcoFlex Kit has a wide array of DNA parts that can be assembled into individual TUs or very large (up to 20!) multi-TU constructs (Moore et al., 2016). The variety of constitutive and inducible promoters, linkers, and tags makes EcoFlex a great option for those interested in recombinant protein purification and metabolic engineering. If instead you want to build genetic circuits and would benefit from a larger library of parts but don’t need to add tags to your proteins, then the CIDAR Kit is an excellent option (Iverson et al., 2016). CIDAR offers a one-pot reaction wherein multi-part assembly can occur. It is quite extensive with a supplemental add-on kit and is compatible with E. coli and/or cell-free systems.

Protein expression in yeast

MoClo is also for eukaryotes! The technology has been used to optimize protein secretion and expression in the biotech workhorse yeast — K. phaffii (formerly known as Pichia pastoris). If your goal is protein isolation, then the MoClo Pichia Kit will get you started (Obst et al., 2017)! It is specifically curated for protein expression in K. phaffii with special attention to protein modifications which can be especially difficult in alternative expression systems such as bacteria.

If your yeast experimental goals are more general in nature, or you aren’t planning on using K. phaffii, then the MoClo YTK Kit specifically designed for S. cerevisiae is at your service (Lee et al., 2015). This kit offers a broad range of parts, including copy number machinery, chromosomal integration modules, and many more of the usual suspects. The Multiplex Yeast Toolkit builds on the YTK and adds several additional capabilities, including conditional gene expression, multiplexed CRISPR, and chromosomal integration of larger expression cassettes (Shaw et al., 2023). Those looking to express high levels of their gene of interest in yeast should also take a look at the Yeast GoldenBraid Cloning System (Pérez-González et al., 2017). In addition to additional elements for S. cerevisiae chromosomal integration and gene expression, the system includes a toolkit to express recombinant proteins within the mitochondrial matrix.

Figure 1: A chart with MoClo kit recommendations for different modal organisms and applications. 

Plant applications

We outlined many synthetic biology MoClo applications, but there are more! Engineering disease resistance through gene stacking, increasing production of a protein of interest, and improving the nutrient profiles within given crops are all possible through MoClo.

The MoClo Plant Parts Kit has everything you would expect in a MoClo kit plus the plant-specific parts you‘ll need, including sub-cellular localization signals, plant markers, and reporters (Engler et al., 2014). This kit is a great starter for your general plant cloning experiments. Addgene also has two additional MoClo plant kits: Plant Parts Kit II and Plant Parts Kit III (Gantner et al., 2018; Chamness et al., 2023). The Plant Parts II kit has a focus on infrastructure with shuttle vectors, several delivery systems which can be engineered into the TUs, and even viral gene silencing vectors. The Plant Parts III kit has a focus on transformation and genome engineering. This is a specialized system with parts including site-specific recombinases, regulatory elements, developmental regulators, and lots of viral components. This kit is also compatible with the Joint Modular Cloning Kit which it builds upon (its ‘basics’ MoClo plant kit buddy). It is important to understand your experimental needs before choosing one or more of these kits, as there are many options!

CRISPR

CRISPR-Cas can harness the power of MoClo to expand both targetability and effectiveness. Perhaps the most intuitive CRISPR MoClo application is to assemble multiple unique sgRNAs into a multi-TU targeting construct (Cas9, sgRNA, reporter, etc.). This allows for the multiplexing of sgRNAs against unique genomic targets or multiple sgRNAs against the same target to ensure efficient cutting. Beyond sgRNAs, MoClo can also be used to test a variety of Cas enzymes with different PAM requirements and editing abilities, such as base editing, nicking, cleaving, etc. The possibilities really are endless: genetic circuits with multiplexed guides, mini screening libraries, direct edit optimization, and more. While there are many CRISPR-Cas libraries and tools available for certain model organisms and backgrounds, MoClo can be invaluable for more complex editing and screening experiments in organisms that don’t yet have these tools built out.

While a generic MoClo system for any given species can be repurposed for CRISPR applications (gene insert = Cas9 + gRNA), Addgene distributes specialized CRISPR kits too. The AspFlex Kit was developed to study the basic biology of and gene regulation in the opportunistic pathogen Acinetobacter baumannii (Brychcy et al., 2023). For plant biologists, Addgene has not one, not two, but three CRISPR MoClo kits for plants that work for both monocots and dicots (Hahn et al., 2020; Chamness et al., 2023; Stuttmann et al., 2021; Grützner et al., 2020). These systems have been used to examine the effects of different promoters, terminators, enhancers, and cross-cassette interactions in gene expression, and to generate multi-gene knockouts.

Mammalian and fly applications

MoClo was initially developed for non-mammalian systems, but it is not confined to that arena! The Mammalian Toolkit was engineered specifically for the types of expression and integration needed in mammalian cell types, and comes with a large library of common parts to get you started quickly (Fonseca et al., 2019). Recently, the toolkit was used to study the effects of protein degradation on chimeric antigen receptor signaling (Kim et al., 2024). The EMMA Toolkit is another great option for mammalian cloning, and is geared towards cloning multi-TU expression constructs (Martella et al., 2017). If you can dream up an experiment, MoClo can help you achieve it!

More recently, a CRISPR MoClo kit termed Fragmid was developed for mammalian systems and Drosophila (McGee et al., 2023). Fragmid developers noticed that studies frequently made comparisons between CRISPR components expressed from different plasmid backbones. They noted that backbone differences such as the order, or distance in the placement of plasmid elements can impact expression levels and consequently convolute the results of these experiments. The modular nature of the Fragmid system allows for direct comparisons of CRISPR parts. The Fragmid system was used to assess how different lentiviral plasmid backbone elements impact viral titer and to compare different Cas enzymes in all-in-one AAV delivery. While Fragmid developers were specifically testing CRISPR tools, it should be easy enough for researchers to expand the Fragmid toolkit to facilitate other lentivirus- and AAV-based studies.

Unified MoClo Systems

While many MoClo kits are designed with specific model organisms in mind, others are designed for flexibility. The Fragmid kit described above works in both mammalian systems and Drosophila. Similarly, the Multi-Kingdom (MK) Golden Gate system was designed to work in fungi, bacteria, protists, plants, and animals (Chiasson et al., 2019). Many plasmid elements such as promoters or fluorophores are interchangeable across organisms. When using the MK system, many of the parts can be used in a variety of organisms, saving time and money for labs with diverse applications. The MK system was used successfully for protein localization and transport studies in HEK cells, recombinant protein purification in bacterial cells, and a two-hybrid protein interaction study in yeast cells.

Need more MoClo? Check out Addgene's MoClo Guide!

Customizing MoClo for your needs

As you probably realize by now, MoClo is a very adaptable system with lots of tools and reagents already in place. MoClo is a fantastic way to save time and money on studies that require many assemblies or that compare the effects of different DNA elements on the behavior of an expression construct. Once you’ve identified your experimental requirements, it’s a good idea to select an available kit that best fits your needs and then modify it as necessary. And remember, if you end up creating a really cool MoClo kit, we would love for you to deposit it with us to share it with the scientific community! Good luck and happy cloning!

This article was written by Susanna Stroik and Meghan Rego, with thanks to Rob Hurt for his expertise and input. 

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References and resources

References

1. Weber, Ernst, et al. “A Modular Cloning System for Standardized Assembly of Multigene Constructs.” PLoS ONE, edited by Jean Peccoud, vol. 6, no. 2, Public Library of Science (PLoS), 18 Feb. 2011, p. e16765. Crossref, https://doi.org/10.1371/journal.pone.0016765.
2. Moore, Simon J., et al. “EcoFlex: A Multifunctional MoClo Kit for E. Coli Synthetic Biology.” ACS Synthetic Biology, vol. 5, no. 10, American Chemical Society (ACS), 2 May 2016, pp. 1059–69. Crossref, https://doi.org/10.1021/acssynbio.6b00031.
3. Iverson, Sonya V., et al. “CIDAR MoClo: Improved MoClo Assembly Standard and New E. Coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology.” ACS Synthetic Biology, vol. 5, no. 1, American Chemical Society (ACS), 4 Nov. 2015, pp. 99–103. Crossref, https://doi.org/10.1021/acssynbio.5b00124.
4. Obst, Ulrike, et al. “A Modular Toolkit for Generating Pichia Pastoris Secretion Libraries.” ACS Synthetic Biology, vol. 6, no. 6, American Chemical Society (ACS), 15 Mar. 2017, pp. 1016–25. Crossref, https://doi.org/10.1021/acssynbio.6b00337.
5. Lee, Michael E., et al. “A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly.” ACS Synthetic Biology, vol. 4, no. 9, American Chemical Society (ACS), 1 May 2015, pp. 975–86. Crossref, https://doi.org/10.1021/sb500366v.
6. Shaw, William M., et al. “A Multiplex MoClo Toolkit for Extensive and Flexible Engineering of Saccharomyces Cerevisiae.” ACS Synthetic Biology, vol. 12, no. 11, American Chemical Society (ACS), 6 Nov. 2023, pp. 3393–405. Crossref, https://doi.org/10.1021/acssynbio.3c00423.
7. Pérez-González, Ana, et al. “Adaptation of the GoldenBraid Modular Cloning System and Creation of a Toolkit for the Expression of Heterologous Proteins in Yeast Mitochondria.” BMC Biotechnology, vol. 17, no. 1, Springer Science and Business Media LLC, 13 Nov. 2017. Crossref, https://doi.org/10.1186/s12896-017-0393-y.
8. Engler, Carola, et al. “A Golden Gate Modular Cloning Toolbox for Plants.” ACS Synthetic Biology, vol. 3, no. 11, American Chemical Society (ACS), 20 Feb. 2014, pp. 839–43. Crossref, https://doi.org/10.1021/sb4001504.
9. Gantner, Johannes, et al. “Peripheral Infrastructure Vectors and an Extended Set of Plant Parts for the Modular Cloning System.” PLOS ONE, edited by Alvaro Galli, vol. 13, no. 5, Public Library of Science (PLoS), 30 May 2018, p. e0197185. Crossref, https://doi.org/10.1371/journal.pone.0197185.
10. Chamness, James C., et al. “An Extensible Vector Toolkit and Parts Library for Advanced Engineering of Plant Genomes.” The Plant Genome, vol. 16, no. 2, Wiley, 9 Mar. 2023. Crossref, https://doi.org/10.1002/tpg2.20312.
11. Brychcy, Merlin, et al. “AspFlex: Molecular Tools to Study Gene Expression and Regulation in Acinetobacter Baumannii.” ACS Synthetic Biology, vol. 12, no. 9, American Chemical Society (ACS), 16 Aug. 2023, pp. 2773–77. Crossref, https://doi.org/10.1021/acssynbio.3c00167.
12. Hahn, Florian, et al. “A Modular Cloning Toolkit for Genome Editing in Plants.” BMC Plant Biology, vol. 20, no. 1, Springer Science and Business Media LLC, 23 Apr. 2020. Crossref, https://doi.org/10.1186/s12870-020-02388-2.
13. Stuttmann, Johannes, et al. “Highly Efficient Multiplex Editing: One‐shot Generation of 8× Nicotiana Benthamiana and 12× Arabidopsis Mutants.” The Plant Journal, vol. 106, no. 1, Wiley, 25 Mar. 2021, pp. 8–22. Crossref, https://doi.org/10.1111/tpj.15197.
14. Grützner, Ramona, et al. “High-Efficiency Genome Editing in Plants Mediated by a Cas9 Gene Containing Multiple Introns.” Plant Communications, vol. 2, no. 2, Elsevier BV, Mar. 2021, p. 100135. Crossref, https://doi.org/10.1016/j.xplc.2020.100135.
15. Fonseca, João Pedro, et al. “A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells.” ACS Synthetic Biology, vol. 8, no. 11, American Chemical Society (ACS), 5 Nov. 2019, pp. 2593–606. Crossref, https://doi.org/10.1021/acssynbio.9b00322.
16. Kim, Matthew S., et al. Degron-Based bioPROTACs for Controlling Signaling in CAR T Cells. Cold Spring Harbor Laboratory, 17 Feb. 2024. Crossref, https://doi.org/10.1101/2024.02.16.580396.
17. Martella, Andrea, et al. “EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid Design and Production of Diverse Expression Vectors.” ACS Synthetic Biology, vol. 6, no. 7, American Chemical Society (ACS), 24 Apr. 2017, pp. 1380–1392. Crossref, doi:10.1021/acssynbio.7b00016.
18. McGee, Abby V., et al. Modular Vector Assembly Enables Rapid Assessment of Emerging CRISPR Technologies. Cold Spring Harbor Laboratory, 27 Oct. 2023. Crossref, https://doi.org/10.1016/j.xgen.2024.100519.
19. Chiasson, David, et al. “A Unified Multi-Kingdom Golden Gate Cloning Platform.” Scientific Reports, vol. 9, no. 1, Springer Science and Business Media LLC, 12 July 2019. Crossref, https://doi.org/10.1038/s41598-019-46171-2.

Resources on Addgene.org

• Modular Cloning (MoClo) Guide
• Addgene’s eBook Collection

More resources on the Addgene blog

• Plasmids 101: Golden Gate Cloning
• High Complexity Golden Gate Cloning with NEB’s DAD
• New MoClo basics blog