Automated Construction of an Open Reading Frame Library from Sinorhizobium meliloti

An automated, high-throughput, open reading frame (ORF) library construction process has been developed. ORFs from genomic DNA of the microbe Sinorhizobium meliloti were amplified by PCR and cloned into the library vector by homologous recombination instead of traditional ligation. From 960 targets, we successfully generated 723 (75.3%) ORFs from the initial PCR. After cloning the successful samples into the library vector, transforming into E. coli and PCR colony screening, 371 (38.6% overall) ORFs were placed into the new library and sequenced. Our prototype library contained 314 (32.7% overall) clones with sequence identity to the Sinorhizobium meliloti genome.