348906 Ex Vivo DNA Assembly

Monday, November 4, 2013
Grand Ballroom B (Hilton)
Laura C. Hayward1, Adam B. Fisher2, Zachary B. Canfield1, Stephen S. Fong1 and George H. McArthur IV1, (1)Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA, (2)Department of Integrated Life Sciences, Virginia Commonwealth University, Richmond, VA

DNA synthesis gives biologists and engineers the ability to program microorganisms by genetically encoding specific functionality.  Although de novo DNA synthesis provides a high level of designability, its application in biology and biotechnology is currently limited due to its cost.  The cost of synthesis does not scale linearly with sequence length and, therefore, it is common for researchers to join short, synthetic DNA molecules into larger constructs using an in-house DNA assembly method. Here we present an inexpensive DNA assembly method utilizing cellular lysates that we call ex vivo DNA assembly.  We tested lysates derived from the common laboratory organisms Escherichia coli and Saccharomyses cerevisiae, and the radio-resistant bacterium Deinococcus radiodurans, for their ability to assemble DNA.  The E. coli-derived lysate was able to facilitate 2- and 3-way assemblies of double stranded DNA fragments, but it was not able to assemble single stranded DNA.  Each assembly was characterized by gel electrophoresis and a novel colorimetric screen in which colonies housing the correctly assembled product expressed a vibrant blue chromoprotein.  This lysate approach for DNA assembly is cheap, fast and reliable, enabling researchers to undertake larger and more complex projects such as constructing combinatorial libraries of a particular metabolic pathway.

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