Protein Engineering Protocols - Mycobacteriology research center

240 Ghadessy and HolligerFig. 1. (A) CSR is based on a simple feedback loop consisting of a polymerase thatreplicates only its own encoding gene. (B) Compartmentalization serves to isolate individualself-replication reactions from each other. (C) In such a system, adaptive gains directly(and proportionally) translate into genetic amplification of the encoding gene, i.e., onlygenes encoding active polymerases (dark gray spheres) are replicated, while inactive variants(light gray hexagons) fail to amplify their own gene and disappear from the gene pool.encoding gene). Compartment integrity must not be compromised during theselection process, which, in the case of polymerases from thermophilic organisms,such as Taq polymerase, includes prolonged exposure to high temperatures.Furthermore, compartments must not allow exchange of macromolecules, suchas DNA or protein (although exchange of small molecules is permissible and, incertain circumstances, desirable). Many different approaches have been describedfor the encapsulation of enzymatic reactions, including lipid vesicles (21). We chose