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