A synthetic phage lambda regulatory circuit

Abstract

Analysis of synthetic gene regulatory circuits can provide insight into circuit behavior and evolution. An alternative approach is to modify a naturally occurring circuit, by using genetic methods to select functional circuits and evolve their properties. We have applied this approach to the circuitry of phage lambda. This phage grows lytically, forms stable lysogens, and can switch from this regulatory state to lytic growth. Genetic selections are available for each behavior. We previously replaced lambda Cro in the intact phage with a module including Lac repressor, whose function is tunable with small molecules, and several cis-acting sites. Here, we have in addition replaced lambda CI repressor with another tunable module, Tet repressor and several cis-acting sites. Tet repressor lacks several important properties of CI, including positive autoregulation and cooperative DNA binding. Using a combinatorial approach, we isolated phage variants with behavior similar to that of WT lambda. These variants grew lytically and formed stable lysogens. Lysogens underwent prophage induction upon addition of a ligand that weakens binding by the Tet repressor. Strikingly, however, addition of a ligand that weakens binding by Lac repressor also induced lysogens. This finding indicates that Lac repressor was present in the lysogens and was necessary for stable lysogeny. Therefore, these isolates had an altered wiring diagram from that of lambda. We speculate that this complexity is needed to compensate for the missing features. Our method is generally useful for making customized gene regulatory circuits whose activity is regulated by small molecules or protein cofactors.

Fig. 1.

Design of TL phage. (A) Representation of the phage λ regulatory circuit. (+) and (−), activation and repression of a promoter, respectively. CIII and CII (not depicted) are expressed from P_L and P_R, respectively. P_L is repressed by CI binding to O_L1 and O_L2. (B) Design of TL phage. The cI and cro genes were replaced with tetR and lacI, respectively. Alleles of lacO were installed in three positions (P_L, O_R3, and P_R), and tetO alleles were installed at two positions (O_L1 and O_R1).

Fig. 2.

Prophage induction by aTc and IPTG. Single lysogens of each variant in strain JL6142 were grown exponentially to 10⁸ cells per ml and treated as described for each panel. (A) Induction by aTc and IPTG. Cultures were exposed to10⁻³ M IPTG (black bar), 10⁻⁷ M aTc (gray bar), or no agent (white bar), shaken 2 h, treated with CHCl₃, and titered. (B) Time course of aTc induction. A lysogen of TL8 was exposed to aTc at 0 (squares), 10⁻¹⁰ M (stars), 10⁻⁹ M (triangles), 10⁻⁸ M (diamonds), or 10⁻⁷ M (circles). Aliquots were treated with CHCl₃ at indicated times and titered. In other experiments (data not shown), 10⁻⁶ M aTc led to the same yield of phage as 10⁻⁷ M aTc. (C) Prophage induction after transient exposure to aTc (triangles) and IPTG (circles). A lysogen of TL21 was exposed to 10⁻⁸ M aTc (triangles) or 10⁻³ M IPTG (circles) at time 0. At the indicated times, aliquots were diluted 1,000-fold, and growth was continued; after 120 min, all samples were treated with CHCl₃ and titered. A TL8 lysogen gave the same kinetics as shown here for TL21 (data not shown), except that the titer at time 0 was higher (also see A).

Fig. 3.

Effect of IPTG on behavior of TL phage. (A) Lysogenization frequency in JL7902 without (white bar) or with 10⁻⁴ M IPTG (gray bar) and 10⁻⁸ M aTc (black bar). (B) Lysogenization frequency (in JL7902) and burst size (in JL6142) of TL10 in an IPTG concentration-shift experiment. Black bar, 10⁻⁷ M IPTG; striped bar, 10⁻⁵ M IPTG.