CHME - Bioseparations Biomaterials Group Project B
Rational de-novo synthesis of genes from synthetic oligonucleotidesWe have constructed a prototype polymerase chain assembly (PCA) workstation which can perform rapid PCA and/or PCR amplification and then separate the full length synthetic dsDNA using tandem immunocapture columns to yield pure synthetic dsDNA (see Figure-1) . Our approach employs rapid gene synthesis by polymerase chain assembly (PCA) in a high speed thermocycler in combination with smart error control strategies to effectively minimize thermal damage [Mamedov et al., J. Biotechnology, 131 (2007) 378-387 and Mamedov et al., Comput Comput Biol. Chem (2008), doi:10.1016/j.compbiolchem.2008.07.021].
For example, endothelial protein C receptor (EPCR, 660 bp) and a mutant EPCR-2 (576 bp) that lacked 4 N-linked glycosylation sites were constructed from 35 and 33 oligonucleotides, respectively, and error rates of less than 1 error/kb were obtained. Further, these synthetic genes were cloned and protein expression was tested in the Pichia pastoris expression system [Mamedov et al., J. Biotechnology, 131 (2007) 387-387]. We are using this technology-tool available in our laboratories in the de-novo assembly of chimeric proteins and customized plasmids. I believe the proposed strategy of rapid de novo gene synthesis – when coupled with proteomics.