Concepts

Introduction

The MoClo standard was first presented in the Weber et al., 2011 21364738 paper, as an attempt to standardize the process of assembling complex DNA molecules from smaller genetic elements. It is inspired by two previous standards:

  • NOMAD 8855278, which proposed generic notions of modules and vectors, as well as assembly using Type IIS enzymes. Modules can be combined in any order, but are clone sequentially one module at a time.

  • BioBrick 18410688, which defines parts with a stable structure: assembling two parts together always gives a part with the same flanking restriction sites.

The MoClo standard enhances both of these assembly standards by relying on the Golden Gate Assembly, which allows single-step assembly of an arbitrary number of modules into a vector. Furthermore, MoClo parts are flanked by stereotypical overhangs, enforcing a particular assembly order, therefore allowing only the desired contruct to be obtained.

Type II-S enzymes

Restriction enzymes are enzymes that are able to cut DNA at or near specific recognition sites. Among those enzymes, Type IIS enzymes cut DNA out of the sequence they recognize, at a defined distance. The cut can produce cohesive ends, which can then recombine with other sequences sharing the complementary cohesive ends, or blunt ends, which cannot recombine. The design of the cohesive ends is of great importance when using Type II-S enzymes to do molecular cloning.

Golden Gate Assembly

The Golden Gate Assembly relies on Type II-S enzymes to assemble several DNA sequences. The sequences are first cut by restriction enzymes, and then assembled together using a T4 DNA ligase. These two steps can be repeated in a single reaction tube using a thermo cycler, as the two enzymes typically do not work at the same temperature. As standard Type II-S enzymes, such as BsaI or BsmBI, create a 4-base-long cohesive end when cutting the DNA, there can be as much as 256 fragments combined together in a deterministic way in a single assembly, although in vivo the chemical properties of the nucleotides will most likely prevent assemblies that large to succeed.

../_images/assembly.svg

Example GoldenGate assembly of two modules in a vector using BsaI.

The MoClo system

The MoClo system combines the idea of a standard part format from the BioBrick standard, with the Golden Gate assembly protocol, allowing several modules to be assembled in a vector at the same time.

Hierarchy

MoClo modules and vectors are divided into several levels, describing their structural and transcriptional features:

  • Level -1 modules are sequences that are not yet in a standardized backbone, but can be assembled in a dedicated vector to form a level 0 module. They are most of the time obtained via oligonucelotide synthesis, or PCR.

  • Level 0 modules are standardized genetic elements: promoter, 5’ UTR, signal sequence, CDS, terminator.

  • Level 1 modules are transcription units, formed by a combination of Level 0 modules, and are able to express proteins

  • Level 2 modules are multigenic units, containing several transcription units, and are able to express many genes at onces.

Furthermore, the enzyme used during the Golden Gate Assembly depends on the assembly level. Alternating between the two enzymes makes it possible for an infinite number of genes to be inserted in the same plasmid, although biological limits are reached in vivo.

Types definition

Although transcription units can be assembled in any possible order in their destination vectors, level 0 modules must be assembled in a specific order to obtain a functional genetic construct. In order to enforce the assembly order, parts are flanked by fusion sites with standard sequences, which are unique to the type of the part. A valid level 1 module is obtained by assembling a part of each type into the destination vector.

Assembly markers

Once the Golden Gate Assembly is finished, the obtained constructs can be amplified using a bacterial host. After transformation, bacteria are selected using two different factors:

  • An antibiotic for which a resistance cassette is only availble on the vector, but not on any module: this allows selecting all the bacterias that received the vector plasmid

  • A marker for a dropout reporter gene that can only be found in the vector but not in the final construct (such as the gfp or lacZ genes).

This double screening makes it possible to select only the bacterias that contain the expected construct, discarding the others, and retrieving the assembled plasmid through a miniprep.

References

8855278

Rebatchouk, D, N Daraselia, and J O Narita. ‘NOMAD: A Versatile Strategy for in Vitro DNA Manipulation Applied to Promoter Analysis and Vector Design.’ Proceedings of the National Academy of Sciences of the United States of America 93, no. 20 (1 October 1996): 10891–96. pmid:8855278

18410688

Shetty, Reshma P, Drew Endy, and Thomas F Knight. ‘Engineering BioBrick Vectors from BioBrick Parts’. Journal of Biological Engineering 2 (14 April 2008): 5. doi:10.1186/1754-1611-2-5

21364738

Weber, Ernst, Carola Engler, Ramona Gruetzner, Stefan Werner, and Sylvestre Marillonnet. ‘A Modular Cloning System for Standardized Assembly of Multigene Constructs’. PLOS ONE 6, no. 2 (18 February 2011): e16765. doi:10.1371/journal.pone.0016765