Engineering complex biological systems in bacteria through recombinase-assisted genome engineering

A protocol enabling the production of biofuels and renewable chemicals via the design and construction of complex biological systems in microbial organisms. Here we describe an advanced paradigm for the design, construction and stable implementation of complex biological systems in microbial organis...

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Veröffentlicht in:Nature protocols 2014-06, Vol.9 (6), p.1320-1336
Hauptverfasser: Santos, Christine Nicole S, Yoshikuni, Yasuo
Format: Artikel
Sprache:eng
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Zusammenfassung:A protocol enabling the production of biofuels and renewable chemicals via the design and construction of complex biological systems in microbial organisms. Here we describe an advanced paradigm for the design, construction and stable implementation of complex biological systems in microbial organisms. This engineering strategy was previously applied to the development of an Escherichia coli –based platform, which enabled the use of brown macroalgae as a feedstock for the production of biofuels and renewable chemicals. In this approach, functional genetic modules are first designed in silico and constructed on a bacterial artificial chromosome (BAC) by using a recombineering-based inchworm extension technique. Stable integration into the recipient chromosome is then mediated through the use of recombinase-assisted genome engineering (RAGE). The flexibility, simplicity and speed of this method enable a comprehensive optimization of several different parameters, including module configuration, strain background, integration locus, gene copy number and intermodule compatibility. This paradigm therefore has the potential to markedly expedite most strain-engineering endeavors. Once a biological system has been designed and constructed on a BAC, its implementation and optimization in a recipient host can be carried out in as little as 1 week.
ISSN:1754-2189
1750-2799
DOI:10.1038/nprot.2014.084