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Golden Gate cloning: how to build a genetic library

Synthetic biology is progressing fast – … However, whilst so many improvements in the field have the potential to revolutionise biotechnology, we maintain a constant limiting factor: our lack of creating a simple, comprehensive method of generating high-quality DNA molecules with multiple genetic elements.

 

So, how exactly can we push synthetic biology over to the next stage? We need a system whereby we can easily select the exact building blocks for genes needed for particular experiments, enabling us to start putting them together. A new molecular cloning system (MoClo), established on the principles of Golden Gate genetic technology, may be able to provide the very system we seek.

 

Let’s take a step back and evaluate how, exactly,’building blocks for genes’ might be selected. Firstly, a generation of a genetic library with different component of genes (differentiating expression and thereby adapting to experimental needs) would need to be compiled. Then, once all gene components are neatly arranged and easily accessible, the next stage of biological synthesis can begin.

 

Imagine designing a car to function exactly as you prefer: you will choose individual parts depending on their function(s), and will need a comprehensive system by which to do so. You will decide which engine to use, which wheels, and which motor. You can modify it depending on what type of car you want to produce, with the individual parts defining the whole vehicle. This is the overall aim of Golden Gate technology: to create constituent parts of genes, fit them together according to how you want the finished product to function, and produce the exact piece of DNA required. Continuing the car analogy, the re-modelling technology first relies on standardising all parts – just like in mass production. Technology is nothing without reliable components with which to construct a final product and, so, through genetic manipulation techniques, it is possible to catalogue constituent parts  of DNA and to mass produce them – after which they can be bound together to generate a final product with the discrete functional element of each component chosen to suit the specific purposes.

 

In principle, engineering DNA as we would a personally designed car sounds perfectly sensible and simple, but in practice is no easy task. Since the overall effect of each gene cannot be predicted from its sequence alone, a challenge arises in engineering the phenotype desired from the number of different building blocks with which each gene can be created. In principle, by systematically sequencing and cataloguing pieces of genes (10-100kb in length), then tweaking the genes by adding or varying the different building blocks (modules), it is possible that they could be used in patterns and combinations to be expressed differently. This understandably takes a number of variations of the constituent parts of the gene, with repeats and a good degree of skill to find the combination of modules offering the optimum result. DNA modules can be selected and bound together to create complex DNA molecules with pieces made up from either natural or artificial DNA.

 

MoClo is not a new technological venture: around 16 years ago, NOMAD, a similar technique, was used to generate another modular like system of gene building blocks. This was further refined with the innovation of Biobrick, in which individual sections of genes could be used. However the most important thing about the Golden Gate system is that not only can you use a number of different fragments in both a sequential/organised manner and in a single reaction step but, unlike NOMAD, you can bind them without the need for superfluous binding regions between the pieces (restriction sites). This allows the DNA pieces to be supplied as uncut DNA rings (plasmids), which can have a mix of restriction and ligation enzymes applied to provide catalysts for the reaction step to join each together. The process can be further enhanced by linking more than one gene together – for example, the writers of this paper linked 11 DNA ringstogether in a three-stage reaction.

Figure 1. General overview of the hierarchical and modular cloning system (showing gene constructs and linking of genes)

Figure 1. General overview of the hierarchical and modular cloning system (showing gene constructs and linking of genes)

 

New synthetic biological technology is of importance in establishing biochemical pathways for metabolic engineering, where  not only do multiple genes need to be targeted for manipulation, but different levels of expression between the genes are required. Since the modules can be standardised and catalogued into modular libraries, technology can be shared between labs, for use by the entire scientific community. It is this innovation that is truly set to revolutionise the field.

Author: Jonathan Hughes/Andrew Jonathan Balmer
Image credit/ doi: 10.1371/journal.pone.0016765
Igen/Freiberg

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One comment on “Golden Gate cloning: how to build a genetic library

  1. Pingback: Gene Cloning | INFOWEBLOG.NET

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