A modular positive feedback-based gene amplifier

Abstract

Background: Positive feedback is a common mechanism used in the regulation of many gene circuits as it can amplify the response to inducers and also generate binary outputs and hysteresis. In the context of electrical circuit design, positive feedback is often considered in the design of amplifiers. Similar approaches, therefore, may be used for the design of amplifiers in synthetic gene circuits with applications, for example, in cell-based sensors.

Results: We developed a modular positive feedback circuit that can function as a genetic signal amplifier, heightening the sensitivity to inducer signals as well as increasing maximum expression levels without the need for an external cofactor. The design utilizes a constitutively active, autoinducer-independent variant of the quorum-sensing regulator LuxR. We experimentally tested the ability of the positive feedback module to separately amplify the output of a one-component tetracycline sensor and a two-component aspartate sensor. In each case, the positive feedback module amplified the response to the respective inducers, both with regards to the dynamic range and sensitivity.

Conclusions: The advantage of our design is that the actual feedback mechanism depends only on a single gene and does not require any other modulation. Furthermore, this circuit can amplify any transcriptional signal, not just one encoded within the circuit or tuned by an external inducer. As our design is modular, it can potentially be used as a component in the design of more complex synthetic gene circuits.

Figure 1

Comparison of constitutive LuxR variants. In these experiments, LuxR was expressed from a tetracycline-inducible promoter, P_LtetO-1, in strain GN101, which harbors a chromosomal copy of tetR. Activity was determined by the ability of these different variants to induce expression from the P_luxI promoter, using GFP as the readout, in the absence of any autoinducer. Dark bars denote the uninduced case and light bars the induced case (200 ng/mL aTc). Error bars denote 95% confidence intervals.

Figure 2

Schematic of positive-feedback amplifier. The basic design for the amplifier consists of GFP and LuxR_Δ2-162 arranged in a bicistronic configuration under the control of the P_luxI promoter. LuxR_Δ2-162 functions in a positive feedback loop as it can bind to the P_luxI promoter and activate its own transcription. In our design, LuxR_Δ2-162 is also used as the input signal for the amplifier. LuxR_Δ2-162, therefore, functions both as the input and positive feedback signal. GFP, the output signal, provides a measure of transcriptional activity.

Figure 3

Schematic of tetracycline sensor coupled to the positive-feedback amplifier. The one-component tetracycline sensor consists of a plasmid where LuxR_Δ2-162 has been cloned behind the TetR-regulated P_LtetO-1 promoter. In the absence of the inducer anhydrotetracycline (aTc), dimeric TetR binds to the O2 operator sites within the P_LtetO-1 promoter and represses transcription. However, when bound with aTc, TetR is no longer able to bind to the O2 operator sites within the promoter, thus enabling dose-dependent control of LuxR_Δ2-162. This sensor was coupled with the positive feedback amplifier, encoded on a separate plasmid, by transforming cells (GN100) constitutively expressing a chromosomal copy of the tetR gene with the two plasmids respectively harboring the sensor and amplifier.

Figure 4

Comparison of tetracycline sensor with positive feedback (solid circles) and without (solid square). Schematic of positive feedback design is shown in Figure 3. The design lacking positive feedback is otherwise identical to one with positive feedback except that only GFP is expressed from the P_luxI promoter. In these experiments, cells were grown overnight at the indicated concentrations of aTc prior to measurements. The fluorescence values were normalized with the OD₆₀₀ absorbance to account for cell density. Error bars denote 95% confidence intervals for the measurement average.

Figure 5

Kinetic analysis of tetracycline sensor with positive feedback (A) and without (B). In these experiments, cells were grown for 12 hours at varying levels of aTc induction with measurements taken every hour. The fluorescence values were normalized with the OD₆₀₀ absorbance to account for cell density. The scale for both sets of experiments is the same. Error bars denote 95% confidence intervals for the measurement average.

Figure 6

Schematic of aspartate sensor coupled to the positive-feedback amplifier. The two-component sensor consists of the Taz sensor kinase and the OmpR response regulator. Taz controls the level of phosphorylated OmpR (OmpR-P), which in turn activates the expression from the P_ompC promoter. When the Taz sensor kinase is bound with aspartate, it increases the levels of OmpR-P, leading to increased expression from the P_ompC promoter. In our design, the Taz sensor kinase has been cloned behind the constitutive P_LtetO-1 promoter on one plasmid (the cells used in these experiments do not possess TetR). On a second plasmid, LuxR_Δ2-162 has been cloned behind the P_ompC promoter, resulting in the expression of LuxR_Δ2-162 being aspartate dependent. The third plasmid harbors the positive feedback amplifier. The sensor was coupled to the amplifier by transforming the three plasmids into a ΔenvZ null mutant (GN101).

Figure 7

Comparison of sensor output in the presence (solid circles) and absence (solid squares) of the positive feedback amplifier. Schematic of positive feedback design is shown in Figure 6. The design lacking positive feedback is otherwise identical to one with positive feedback except that only GFP is expressed from the P_luxI promoter. In these experiments, cells were grown overnight at the indicated concentrations of aspartate prior to measurements. The fluorescence values were normalized with the OD₆₀₀ absorbance to account for cell density. Inset figure shows the magnification of the response for the design lacking positive feedback. Error bars denote 95% confidence intervals for measured averages.