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Team:Lethbridge CCS/Project
From 2008.igem.org
Our project centers on the development and optimization of a ligase-independent cloning (LIC) protocol.
Abstract
Ligase-Independent Cloning as a Standard for BioBrick Preparation
While there is an established BioBrick format, there is not yet a standard method for turning a gene of interest into a BioBrick. Ideally, such a standard method would be easily adopted, even by amateurs, and would lend itself to automation. A significant drawback of several existing techniques is their dependence on ligase treatment, which is often problematic. We propose a ligase-independent cloning (LIC) method, based on the technique of [http://nar.oxfordjournals.org/cgi/content/abstract/18/20/6069 Aslanidis & de Jong (1990)], as a possible standard for novel BioBrick preparation. Instead of short overhangs and ligase treatment, LIC uses long overhangs to circularize plasmid vectors for transformation without the use of ligase. The LIC method reduces the number of enzyme steps required for cloning, thus lending itself to easy adoption, automation, and real biological 'engineering.'
Rationale
The underlying idea of iGEM is that an engineering approach can be applied to biology. Using a computer analogy, the goal is to have software (DNA sequences) that can be run on compatible hardware (microorganisms) anywhere in the world.
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Ideally, this software-hardware interface would be highly portable, but also simple. The BioBricks used by iGEM are advertised with 'Lego' blocks, implying a very easy-to-use set of tools. Our team of high school students discovered that (at this stage, at least) synthetic biology is NOT yet at the stage of quick and easy implementation in just anyone's kitchen or garage.
Also, the standards that are used do not apply to all components of the process. While there are standards for the structure of a BioBrick, and for how existing BioBricks are to be composed, there is not a single method for turning interesting DNA sequences into BioBricks.
Given the above, we chose to investigate ligase-independent cloning, a technique we believe can fill in a gap in the standards for iGEM, in a way that is relatively easy to implement and that also lends itself to automation in the future.
Plasmid: [http://partsregistry.org/Part:BBa_K155000 K155000]
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