Programmable cells: interfacing natural and engineered gene networks

Abstract

Novel cellular behaviors and characteristics can be obtained by coupling engineered gene networks to the cell's natural regulatory circuitry through appropriately designed input and output interfaces. Here, we demonstrate how an engineered genetic circuit can be used to construct cells that respond to biological signals in a predetermined and programmable fashion. We employ a modular design strategy to create Escherichia coli strains where a genetic toggle switch is interfaced with: (i) the SOS signaling pathway responding to DNA damage, and (ii) a transgenic quorum sensing signaling pathway from Vibrio fischeri. The genetic toggle switch endows these strains with binary response dynamics and an epigenetic inheritance that supports a persistent phenotypic alteration in response to transient signals. These features are exploited to engineer cells that form biofilms in response to DNA-damaging agents and cells that activate protein synthesis when the cell population reaches a critical density. Our work represents a step toward the development of "plug-and-play" genetic circuitry that can be used to create cells with programmable behaviors.

Fig. 1.

The modular structure of a simple programmable cell.

Fig. 2.

Transitions in the genetic toggle switch. (A) The network has two stable expression states (indicated by gray boxes) where λ CI represses lacI expression and LacR represses λ cI expression, respectively. Transition from the high λ CI state can be induced by degrading λ CI or by introducing additional LacR molecules. (B) Simulated transition induced when the rate of λ CI proteolysis is temporarily increased under inducing conditions.

Fig. 3.

Interfacing the SOS signaling pathway in strain A1. (A) Diagram of the engineered genetic circuitry. The genetic toggle switch module (pTSMa) controls the expression of GFP from plasmid pCIRb in response to DNA damage. (B) Induction of GFP expression after exposure to MMC. (C) Induction of GFP expression after 1-10 s of UV irradiation.

Fig. 4.

Example of programmed phenotype in strain A2. (A) Diagram of the engineered genetic circuitry. The genetic toggle switch module (pTSMa) controls the expression of traA from plasmid pBFR in response to DNA damage. (B) Biofilm formation quantified by crystal violet staining in cultures of strain K12/AK4 (positive control), strain K12/AK3 (negative control), and strain A2 (programmed E. coli). (C and D) Pictures of microfermentors incubated with untreated cells (C) or cells treated with MMC (D).

Fig. 5.

Interfacing an AHL biosensor module in strain B1. (A) Diagram of the engineered genetic circuitry. (B) Repeated activation and deactivation of GFP expression by using IPTG and AHL, respectively. Cultures were induced for 12 h, as indicated, followed by growth for 12 h without inducers. (C) Population-averaged fluorescence signal in the presence of AHL. The cell population is partially induced (bimodal response, see Inset) at intermediate AHL concentrations. Open and closed circles in B and C indicate fluorescence measured in populations initially grown at 42°C and treated with IPTG, respectively.

Fig. 6.

Density-dependent gene activation in strain B2. (A) Diagram of the engineered genetic circuitry. The luxI and luxR genes are expressed constitutively. (B) Cell density-dependent expression of GFP. (C) The expression of GFP in cells that lack luxI and are unable to produce AHL.