What is Modular Cloning?
The production of medication or vaccines in genetically modified organisms is no longer a futuristic vision. It has been part of our lives for quite a while now and will become an even bigger part in the next few decades. But not only regarding medical sciences. Synthetic biology offers more and more possibilities in other completely different fields. For example the manufacturing of biofuels or cosmetics in these so called GMOs will also become a bigger part in our daily live. The list of possible applications seems endless.
However, genetic engineering and the introduction of new metabolic pathways into an organism is extremely time-consuming and labour-intensive. Therefore scientists try to develop better methods in order to make their own work much easier.
One of the most modern and most efficient methods in Cloning is called “Modular Cloning”, or “MoClo”. This building-block-system developed by Ernst Weber et al.[1] allows simple assembly of different DNA-building blocks, resulting in a fully functional gene. The gene can then be introduced into an organism, either individually or in combination with other genes, to serve its purpose.
LEGO for Biologists
Thanks to the modularity of this system, the individual parts of the gene, e.g. promotor, signal peptides and coding regions, can be assembled in a way similar to LEGO bricks.
The basics
Modular cloning is based on a system called Golden Gate Assembly, which was established by Marillonet et al. in 2008.[2] The system uses so called type IIS restriction enzymes, which cleave DNA at a specific position. The important feature of those specific Type IIS – enzymes is the fact that they do not cut DNA inside their specific recognition site but rather in a predetermined distance to that. The result is a four nucleotide overhang on both DNA-strands, which are called “sticky ends”.
DNA-Scissors
Type IIS restriction enzymes (e.g. BsaI) recognise specific DNA-sequences but cleave at a different position with a predetermined distance to the recognition site.
In modular cloning, this ability is useful for two very important aspects. After designing the module that is later supposed to become a part of the gene (e.g. a promotor), the following step is the treatment with the restriction enzyme. As a result of the recognition site not being equal to the cleaving site, it is possible to simply cut off the recognition site if the enzyme is used correctly. This results in a product which does not carry the recognition site anymore.
In short: Once incorporated into the right place, it cannot be removed by the same restriction enzyme.
The advantage of this process: Cleaving of the designed module and incorporation into the destination vector can take place simultaneously in one reaction tube and the reaction is massively forced in the direction of the desired product.
The second aspect is what makes modular cloning modular. It is the ability of the restriction enzyme to always cut in the same distance to the recognition site. If the distance between the sites and the length of the nucleotide overhang is known, one can basically choose which nucleotides the overhang should contain.
But why is this important? When choosing those overhangs wisely and fitting to one another, the modules will always assemble in the correct order.
The advantage of the whole process: When establishing the system for a specific organism, the nucleotide overhangs and thereby the transitions between modules are standardised. After finishing the module library it is possible to pick the individual gene-parts and because of the standardised transitions they will always assemble in the correct order.
Apart from that, the module-library can later be extended by scientists all around the world, as long as the module-transitions are used correctly.
Examples
One example for the already very successful establishment of this system is the microalgae Chlamydomonas reinhardtii. Here, modular cloning was already established in 2018 by an international group of scientists. At the time of publication, the library already contained 119 modules with different functions.
Chlamydomonas reinhardtii
The microalgae is one of the organisms in which modular cloning already found a home. One big advantage of the algae: The energy for all of its metabolic pathways is produced through photosynthesis and therefore it does not need extra “feeding”.
Sources
[1] 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; PMCID: PMC3041749.
[2] Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability. PLoS One. 2008;3(11):e3647. doi: 10.1371/journal.pone.0003647. Epub 2008 Nov 5. PMID: 18985154; PMCID: PMC2574415.
[3] Crozet P, Navarro FJ, Willmund F, Mehrshahi P, Bakowski K, Lauersen KJ, Pérez-Pérez ME, Auroy P, Gorchs Rovira A, Sauret-Gueto S, Niemeyer J, Spaniol B, Theis J, Trösch R, Westrich LD, Vavitsas K, Baier T, Hübner W, de Carpentier F, Cassarini M, Danon A, Henri J, Marchand CH, de Mia M, Sarkissian K, Baulcombe DC, Peltier G, Crespo JL, Kruse O, Jensen PE, Schroda M, Smith AG, Lemaire SD. Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synth Biol. 2018 Sep 21;7(9):2074-2086. doi: 10.1021/acssynbio.8b00251. Epub 2018 Sep 5. PMID: 30165733.
Images
CHLAMYDOMONAS REINHARDTII: Photo by Y. Tsukii, http://protist.i.hosei.ac.jp/pdb/images/chlorophyta/chlamydomonas/Euchlamydomonas/reinhardtii/sp_10.html