has relied on a universal toolbox containing three indis-pensable implements: restriction enzymes, ligases and, last but not least, Escherichia coli. Even those of us who now languish behind laptops and in meetings rather than work at the bench instantaneously recognize the smells of Luria broth and plasmid preps. Since the hallmark paper of Cohen and colleagues (1973), many elegant variations on the theme of restriction and ligation have been introduced to make E. coli-based cloning tremendously versatile. However, especially in the field of metabolic engineering, two major limitations of this trusted approach gradually became apparent. First, as the complexity of the desired DNA constructs increased, design of multi-gene constructs based on restriction and ligation turned into a molecula
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