Generating multigene constructs with the GoldenGate-based MoClo cloning system.
Sylvestre Marillonnet (IPB) together with Ramona Grützner (IPB) and Stefan Werner (Nambawan Biotech GmbH, Halle) has recently published two method chapters in the "DNA Cloning and Assembly" volume of the popular Methods in Molecular Biology book series. One chapter deals with generation of MoClo standard parts using Golden Gate Cloning (Grützner and Marillonnet, 2020) and the other chapter covers assembly of multigene constructs using the MoClo modular cloning system (Marillonnet and Werner, 2020).
Availability of efficient DNA assembly methods is a basic requirement for synthetic biology. Based on the Golden Gate cloning procedure for DNA assembly, the MoClo modular cloning method enables users to assemble multigene constructs from basic parts like promoters, coding sequences, or terminators, using a series of successive one-pot assembly reactions.
In the Grützner and Marillonnet, 2020, method chapter, the authors provide a detailed protocol for cloning of basic parts. All steps from first defining the type of basic part that needs to be cloned, primer design, and fragment amplification to finally Golden Gate cloning and sequencing are described. Also, the authors address cloning of subparts to generate basic parts that are larger in size. As a result, basic parts are at hand for further assembly steps.
The Marillonnet and Werner, 2020, method chapter provides instructions for performing these subsequent steps for generating multigene constructs using the modular cloning system MoClo. The protocol guides the experimenter through preparing the construction strategy and lays out a set of standard rules to be followed for the assembly strategy. Using a series of one-pot reactions with the set of identified parts and vectors, the multigene constructs can then be assembled.
Modular cloning systems like MoClo have many advantages for complex pathway engineering. These systems are simple to use, efficient, and allow users to assemble multigene constructs starting from libraries of cloned and sequenced parts. The efficiency of these systems is especially useful when generating libraries of numerous construct variants.
Original publications:
Grützner R., Marillonnet S. (2020) Generation of MoClo Standard Parts Using Golden Gate Cloning. In: Chandran S., George K. (eds) DNA Cloning and Assembly. Methods in Molecular Biology, vol 2205. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0908-8_7
Marillonnet S., Werner S. (2020) Assembly of Multigene Constructs Using the Modular Cloning System MoClo. In: Chandran S., George K. (eds) DNA Cloning and Assembly. Methods in Molecular Biology, vol 2205. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0908-8_8