Synthetic Biology Enables Programmable Cell-Based Biosensors

Abstract

Cell-based biosensors offer cheap, portable and simple methods of detecting molecules of interest but have yet to be truly adopted commercially. Issues with their performance and specificity initially slowed the development of cell-based biosensors. With the development of rational approaches to tune response curves, the performance of biosensors has rapidly improved and there are now many biosensors capable of sensing with the required performance. This has stimulated an increased interest in biosensors and their commercial potential. However the reliability, long term stability and biosecurity of these sensors are still barriers to commercial application and public acceptance. Research into overcoming these issues remains active. Here we present the state-of-the-art tools offered by synthetic biology to allow construction of cell-based biosensors with customisable performance to meet the real world requirements in terms of sensitivity and dynamic range and discuss the research progress to overcome the challenges in terms of the sensor stability and biosecurity fears.

Keywords: cell-based biosensor; genetic circuits; rational approaches; response curve; synthetic biology.

Figure 1

Biosensor architecture: A cell‐based biosensor works by the input entering the cell being detected by the sensor component (sensor) of the genetic circuit which is a gene constitutively expressing a transcription factor (TF) which then activates the generation of a reporter protein (output) by binding and activating the promoter responsive to the transcription factor (P_TF).

Figure 2

Bacterial two‐component systems for biosensing: a) The sensor module of the TCS sensor kinase binds to the target resulting in activation of the kinase domain, the response regulator is then phosphorylated by the activated kinase domain to activate the response regulator. The response regulator will then bind to the output promoter (P_R) to generate the response. b) Response regulators containing their cognate receiver domain (RD) and DNA binding domain (DBD) will bind to their cognate output promoter (P_R), response regulators with swapped DBD will then bind to an alternative output promoter (P_S) which generates a stronger output.47

Figure 3

Approaches for universal cell‐based biosensor platforms: Universal biosensors aim to link specific recognition elements to a generic platform to expand the range of target molecules capable of being sensed through cell‐based biosensors, whilst generating a system that allows quick construction and optimisation of new cell‐based biosensors. a) Split protein biosensor systems work by adding the ligand binding domain (LBD) to each separate half of a protein which when brought back together generates a response. i) Split T7 polymerase (Split T7) is used to activate expression of a reporter protein from the T7 promoter (P_T7).48 ii) Split glucose dehydrogenase is used to control the degradation of glucose releasing electrons which can be detected through a range of methods.49 b) The target molecule is broken down using a natural metabolic pathway (metabolic transducer) into a metabolic product which can be detected by a transcription factor to activate expression of a reporter from a responsive promoter (P_R).51

Figure 4

Characteristics of a typical biosensor response curve: The dynamic range is ratio between the minimum and maximum output expression ($max/min$ ). The limit of detection (LOD) is the lowest concentration of the target which can be detected from the background response. The leaky expression is the level of reporter present when no target is present. These characteristics are commonly used to define the response of the biosensor. The operating range gives the concentrations of target which can be detected through a change in the output.

Figure 5

Tuning biosensor response through optimisation of intracellular receptor densities: a) By altering the intracellular concentration of the ligand‐responsive allosteric transcription factor (TF) the output from the response promoter (P_R) can be altered. i) For activators, increasing the concentration of transcription factor can increase output from the response promoter making it more sensitive to the target molecule. ii) For repressors reducing the intracellular concentration of the transcription factor can increase the output from the response promoter making it more sensitive to the target molecule.56, 57 b) Altering the intracellular concentration of the target molecule can also be used to alter the response of the biosensor. The intracellular concentration can be increased by engineering import machinery to increase transport into the cell to increase the response.26

Figure 6

Part engineering to optimise dynamic range: a) Anderson collection of promoters (http://parts.igem.org/Promoters/Catalog/Anderson) are well characterised constitutive promoter sequences of a range of strengths which could be used to replace the −35 and −10 sites of responsive promoters to alter their behaviour. Different strength promoter sequences will alter the maximum expression and the leakiness. Response curves are shown for activator and repressor systems.59 b) Alternative ribosome binding site (RBS) sequences of different strengths can be replaced to alter the response of the biosensors.60, 61 The response curves for activator and repressor systems with different RBS sequences are shown.

Figure 7

Biological signal amplification to boost dynamic range: Genetic amplifiers are ligand‐free ultrasensitive transcription factor and promoter pairs which have a large output dynamic range and can be linked to sensing transcription factors and promoters with low dynamic ranges to increase them. a) The simplest architecture of an amplifier is an activator system with high expression where the responsive promoter (P_R) is used to control the expression of the activator (hrpS and hrpR) which then activates the amplifier promoter (P_hrpL). The effect of an amplifier on the response curve is shown by the two curves. The level of amplification (amplification gain) can be tuned by adding another level of regulation to the amplifier through a repressor (hrpV).63 b) Alternative architectures can also be used for amplification. i) Positive feedback can be added to increase the amplification on induction placing the amplifier protein (amp) under the control of the amplifier promoter (P_amp) and the responsive promoter (P_R).64 ii) Amplifiers have been combined with other techniques to prevent increased leaky expression due to amplification.1 The addition of a degradation tag to the output protein (P_arsR) reduces basal expression. Whilst the expression of a protease under the control of the same input promoter can cleave off the degradation tag when the target is present to prevent reduction in the maximal expression.

Figure 8

Approaches to reduce leaky expression: a) Degradation tags (Deg tags) can be used for post‐translational reduction of leaky expression. Degradation tags increase the degradation of the reporter protein and reduce the levels within the cells. The strength of the degradation tag will determine the reduction in the levels of the reporter protein.8, 61 The two curves show the change in the response curve with a degradation tag (+ Deg tag) or without (− Deg tag) for activators and repressors. b) A downstream promoter placed on the non‐coding strand can be used to produce a complementary RNA to the mRNA to generate a double stranded complex which is degraded or blocks the ribosome to prevent translation. The level of mRNA prevented from being used in translation is controlled by the level of complementary RNA which can be altered by changing the strength of the promoter used to express the complementary RNA.66 The two curves show the change in the response curve with antisense transcription (+ Antisense transcription) or without (− Antisense transcription) for activators and repressors. c) Decoy sites titrate the binding of the transcription factor away from the responsive promoter (P_R). For activator systems this can reduce leakiness by preventing binding of the transcription factor in the absence of target.8, 67 Whilst for repressors this can be used to reduce the amount of effective repressor. The two curves show the change in the response curve with decoy sites (+ Decoy sites) or without (− Decoy sites) for activators and repressors. d) For repressors the position and number of operator sites can be altered to improve the efficiency of the repressor to reduce leaky expression. i) There are three possible sites for the where the operator can be. First the core site (C) is most efficient and its efficiency is increased when the binding site overlaps with either the −35 or −10 regions.59 Second is the proximal site (Pro) downstream of the transcription start site where the repressor can act as a physical block to the polymerase followed by the distal site (Dis).68 ii) If the initial operator site (Op) cannot provide enough repression then an additional site can be added downstream to act as a physical ‘roadblock’.69

Figure 9

Integrating multiple responsive promoters to increase sensing selectivity: An AND logic gate can be used to generate a highly selective biosensor for zinc. Each zinc responsive promoter responds to another metal ion, promoter P_zraP also responds to lead and the promoter P_zntA also responds to cadmium. The output promoter P_hrpL is activated by a protein complex formed from two different subunits (HrpR and HrpS). By splitting the expression of these two components under the control of different target responsive promoters (P_zraP and P_zntA) this ensures that reporter (GFP) expression only occurs when both promoters are activated. Activation of both promoters will only occur in the presence of zinc and not with either of the non‐specific molecules (lead and cadmium).28