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. Author manuscript; available in PMC: 2014 Mar 15.
Published in final edited form as: Methods. 2012 Mar 23;60(1):81–90. doi: 10.1016/j.ymeth.2012.03.009

Figure 3.

Figure 3

Network level directed evolution strategies. (a) Directed evolution of the LuxR transcriptional regulator has been achieved through screening, ON selection, or OFF selection, where active LuxR stimulates transcription of a GFP marker, a chloramphenicol resistance gene, or a β-lactamase inhibitor, respectively. (b) Single gene selection markers such as hsvTK allow selection of both ON and OFF states. In ON selection, exogenously supplied 2’-deoxy-5-fluorouridine (5FdU) is converted by the host to 2’-deoxy-5-fluorouridine monophosphate (5FdUMP), which blocks de novo dTMP biosynthesis. Expression of hsvTK activates dTMP biosynthesis via the salvage pathway, rescuing the host. In OFF selection, leaky hsvTK expression produces the mutagenic nucleotide monophosphate dPMP from exogenously supplied dP, leading to host cell death.