Regulatory circuit design and evolution using phage lambda

Abstract

Bistable gene regulatory circuits can adopt more than one stable epigenetic state. To understand how natural circuits have this and other systems properties, several groups have designed regulatory circuits de novo. Here we describe an alternative approach. We have modified an existing bistable circuit, that of phage lambda. With this approach, we used powerful genetic selections to identify functional circuits and selected for variants with altered behavior. The lambda circuit involves two antagonistic repressors, CI and Cro. We replaced lambda Cro with a module that included Lac repressor and several lac operators. Using a combinatorial approach, we isolated variants with different types of regulatory behavior. Several resembled wild-type lambda--they could grow lytically, could form highly stable lysogens, and carried out prophage induction. Another variant could form stable lysogens in the presence of a ligand for Lac repressor but switched to the lytic state when the ligand was removed. Several isolates evolved toward a desired behavior under selective pressure. These results strongly support the idea that complex circuits can arise during the course of evolution by a combination of simpler regulatory modules. They also underscore the advantages of modifying a natural circuit as an approach to understanding circuit design, systems behavior, and circuit evolution.

Figure 1.

Design of λlacI. (A) Logical representation of the phage λ lysis–lysogeny circuit. Plus (+) and minus (-) signs indicate activation and repression of promoter, respectively (Lac repressor could repress P_RM when a lacO variant was in the position of O_R3). CIII and CII (not shown) are expressed from P_L and P_R, respectively. Expression of P_L is controlled in λ by CI binding to two operators, O_L1 and O_L2, not shown here, which lie to the right of P_L in positions analogous to O_R1 and O_R2, respectively. (B) Control of regulatory state by the pattern of occupancy by CI or Cro. The circuit is maintained by the actions of CI and Cro at the O_R region. CI stabilizes the lysogenic state, and Cro stabilizes the lytic state. (C) Design of λlacI. Cro was replaced with lacI. LacO alleles were installed in two positions (P_L and P_R); at the position of O_R3, some variants had a lacO allele, and some had O_R3. The same set of lacO alleles was used at all three sites. (D) LacO alleles and the Shine-Dalgarno sequences used in combinatorial approach. Allele A is wild-type lacO; mutations in other lacO alleles are in bold type; binding is progressively weaker going from B to E (Betz et al. 1986). W is λ O_R3. In box SD, Shine-Dalgarno sequences and start codons are in bold type; SD alleles allow progressively less-efficient translation going from A to F (Gardner et al. 2000). (E) O_R3 and lacO are in bold type. O_R1 and O_R2 are italicized. Locations of -35 and -10 regions for the promoters are shown. The base substitutions at the ends of the lacO at O_R3 should not affect LacI binding (Betz et al. 1986).

Figure 2.

UV dose responses for prophage induction. Exponentially growing cultures of lysogens were centrifuged, suspended in TMG, and irradiated (see Materials and Methods) at the indicated doses; aliquots were diluted 10-fold in LBGM and shaken for 2 h at 37 °C in the dark, treated with CHCl₃, and titered. (Crosses) Wild type; (diamonds) AWCF; (stars) AWBF; (squares) AWCE; (triangles) AWBE.

Figure 3.

Time course for cell growth and free phage release by lysogens of the wild type and CECA, with or without IPTG. Lysogens of wild type and CECA in strain JL7223 were isolated by kanamycin selection, and single lysogens were identified by a PCR test (Powell et al. 1994); those of CECA were propagated with 10^-4 M IPTG. For both strains, overnight cultures were grown in LBGM with 10^-4 M IPTG and grown to exponential phase in the same medium; after washing by centrifugation, portions were diluted and grown with or without IPTG. Aliquots were removed at the indicated times; cell number was estimated by O.D.₅₉₀ (A), and free phage titers (B) were measured. (Open circles) Wild type without IPTG; (closed circles) wild type with IPTG; (open squares) CECA without IPTG; (closed squares) CECA with IPTG. In A, phage/cell is not shown for 300 or 330 min, because the O.D.₅₉₀ does not give an accurate cell count in the presence of cell debris from lysis; phage titers at 270, 300, and 330 min were 4 × 10⁸, 9 × 10⁸, and 2 × 10⁹/mL, respectively.