We have constructed a modular AND gate based on the amber suppression of T7 RNA polymerase. Only when two input promoters are active is an output turned on. Because the inputs and outputs of this gate are transcriptional signals, they can be easily replaced. This modularity is demonstrated by swapping the inputs and outputs of the circuit while preserving the AND-gate behavior. In this paradigm, changes in transcription become a common currency allowing the modular integration of individual devices (Weiss et al, 1999
; Endy, 2005
The modular nature of this type of transcriptional logic gate will facilitate its use in a variety of engineering applications. In particular, AND logic would be useful in obtaining gene expression in a specific microenvironment. A set of promoters—identified rationally or with a microarray—could be used as inputs to the AND gate. Rather than directly detecting the presence of a new environment with a single engineered promoter or sensing system, independent promoters sense different aspects of the environment. The AND gate activates only when all conditions are present to induce cellular responses. Often microenvironments are defined by multiple nonspecific signals such as oxygen, pH, cell density, lactate, and glucose. It is only when several of these inputs are integrated that specificity can be achieved.
A two-dimensional transfer function of a simple mathematical form captures the input–output behavior of the AND gate. This model could be used to predict the genetic changes required to connect two inputs to the circuit to produce a functional AND gate. To be able to use the model, the inputs have to be characterized using the plasmid and fluorescent reporter system used in this study. In this sense, this work represents a step toward standardization, where circuits are characterized quantitatively to understand their collective function when connected in series. This will be a critical approach in the design of large integrated systems consisting of multiple genetic circuits.
The transfer function we derived relies on a steady-state assumption and is based on a deterministic model. However, the response of the circuit may have dynamic or stochastic aspects that are not predicted from the model. For some applications where the induction of inputs is transient, the dynamics of the circuit could be critical to the successful implementation of the output process. A further obstacle to standardization is that the circuit may show different response characteristics in different environments or stages of growth. For example, this AND gate produces a lower gain at low cell densities (not shown). This change in the output range could impact its connection to a downstream circuit.
The ultimate goal in genetic circuit design is to incorporate them into more complex systems consisting of multiple circuits, sensors, and actuators. Unlike electronic circuits, where the spatial wiring of a circuit determines the flow of information through the circuit, intracellular circuits prevent cross-communication through specificity of biochemical interactions. Once a part—such as T7 or SupD—is used, it cannot be used in any of the other devices. Therefore, the use of the T7 RNA polymerase-based gate makes this valuable gene unavailable for protein overexpression within the same system. An advantage of our design is that any particular transcriptional activator, including engineered sequence-specific transcription factors (Mandell and Barbas, 2006
), could be used in place of the polymerase gene. Similarly, the use of amber suppression precludes its use in other systems. For example, translational recoding with unnatural amino acids using amber suppression could not coexist with this logic gate (Wang et al, 2006
). Other translational regulators that could be used in this gate include nonsense, missense, and frameshift suppressors, or riboregulators (Anderson et al, 2002
; Isaacs et al, 2004
Pushing the boundary of genetic engineering will require a toolbox of genetic circuits that perform prescribed functions and are designed to be incorporated into larger systems. Toward this end, we have described the construction and analysis of a genetic AND gate. We have demonstrated that this gate is modular, so that it can be connected to different promoter-based inputs and used to drive different outputs. Further, in a step toward standardization, we developed a model that could be empirically parameterized, and used to predict how new promoters will connect to the gate. Circuits like this will find broad application in genetic engineering of systems in which multiple transcriptional signals must be combined to produce a specific cellular response.