Mol Syst Biol. 3: 134 A paradigm shift is underway within the methodology of heterologous protein expression. Specifically, researchers are moving away from conventional techniques of cloning genes from cDNA libraries and moving toward the rational design and de novo synthesis of entire protein‐coding sequences from pre‐annealed oligonucleotides (Libertini and Di Donato, 1992; Gustafsson et al , 2004). It was the invention of polymerase chain reaction (PCR) that allowed efficient construction of synthetic genes. Since then, the steadily increasing accuracy and decreasing cost of oligonucleotide synthesis (now as low as $0.10 per base; Carlson, 2003; Carr et al , 2004; Kong et al , 2007, see Figure 1) has created a research environment in which gene synthesis offers three main advantages over molecular cloning: cost efficiency, scope and flexibility of redesign (Libertini and Di Donato, 1992). As a result, the emerging field of synthetic biology is highly motivated to improve this approach, as it seeks to expand the sophistication of human‐engineered genetic architectures, leading ultimately to the synthesis of entire genomes (Yount et al , 2000; Smith et al , 2003). Figure 1. The cost, per base, of commercial oligonucleotide assembly from 1999 to 2006. The price of gene synthesis has decreased almost 30‐fold in the past 7 years (data for years 1999–2003 are taken from Carlson, 2003). Data for years 2004–2006 reflect the lowest price found in advertisements placed within Science magazine). Current research into synthetic gene construction has focused largely on improving PCR‐based methods. Areas under active investigation include the following: increasing the accuracy of gene products by reducing errors in oligonucleotide construction and PCR synthesis/amplification (Ciccarelli et al , 1991; Young and Dong, 2004), reducing the relatively high cost of post‐synthesis sequencing (Young and Dong, 2004), increasing the length of genes …