282680 Modular Production and Cloning of Error-Prone Genomic Libraries

Wednesday, October 31, 2012
Hall B (Convention Center )
Benjamin G. Freedman and Ryan S. Senger, Biological Systems Engineering, Virginia Tech, Blacksburg, VA

A genomic library produced from cloning and over-expressing sheared DNA is an important tool in combinatorial metabolic engineering.  This technique has led to several improved phenotypes in response to environmental stress, and efforts have been implemented to enrich genomic libraries to optimize traits such as product secretion that are not tied to growth.  However, traditional methods of DNA shearing, polishing, and blunt-end cloning are highly inefficient and time-consuming.  Newer TA-cloning methods provide improvements but are also far from ideal.  Here, we present a method for generating genomic libraries through random priming PCR and modular Gibson-inspired cloning method that allows extremely high (i) control over product insert size and (ii) cloning efficiency.  This method is also capable of generating non-native fragments through error-prone PCR methods.  We demonstrate these methods in detail and provide examples of library enrichment leading to new strains of Clostridium cellulolyticum optimized for cellulose consumption in consolidated bioprocessing conditions. 

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