Layering genetic circuits to build a single cell, bacterial half adder

Gene regulation in biological systems is impacted by the cellular and genetic context-dependent effects of the biological parts which comprise the circuit. Here, we have sought to elucidate the limitations of engineering biology from an architectural point of view, with the aim of compiling a set of engineering solutions for overcoming failure modes during the development of complex, synthetic genetic circuits.
Results

Using a synthetic biology approach that is supported by computational modelling and rigorous characterisation, AND, OR and NOT biological logic gates were layered in both parallel and serial arrangements to generate a repertoire of Boolean operations that include NIMPLY, XOR, half adder and half subtractor logics in a single cell. Subsequent evaluation of these near-digital biological systems revealed critical design pitfalls that triggered genetic context-dependent effects, including 5? UTR interferences and uncontrolled switch-on behaviour of the supercoiled ?54 promoter. In particular, the presence of seven consecutive hairpins immediately downstream of the promoter transcription start site severely impeded gene expression.
Conclusions

As synthetic biology moves forward with greater focus on scaling the complexity of engineered genetic circuits, studies which thoroughly evaluate failure modes and engineering solutions will serve as important references for future design and development of synthetic biological systems. This work describes a representative case study for the debugging of genetic context-dependent effects through principles elucidated herein, thereby providing a rational design framework to integrate multiple genetic circuits in a single prokaryotic cell.
Background

Gene regulation in biological systems behaves like a molecular computer whereby the gene’s output can be modelled as on-off states of Boolean (digital) logic [1, 2, 3]. However, programming gene regulation is far from trivial and requires considerable time and effort during functional testing and tuning of the synthetic genetic circuits under development. Apart from the scarcity of reliable and well-characterised biological parts, digital performance in biological systems is further impacted by the cellular and genetic context-dependent effects of the biological parts which comprise the circuit [4, 5, 6]. Recent studies have shown that genetic crosstalk between the engineered circuits and endogenous networks of the host cell can lead to cellular context-dependent effects [7, 8]. For this reason, molecular parts and devices that are orthogonal to the cell native machineries with roles in either genetic transcription or protein translation have been created to enable predictable engineering of genetic circuits [9, 10, 11, 12, 13]. Demonstrations of layered genetic circuits in a single cell, such as the execution of a 4-input AND gate in bacteria [10] and biological half adders and half subtractors in mammalian cells [14] have revealed that orthogonal logic gates can be interlinked to perform digital operations of higher complexity and diversified outputs. While the capability to program cells with memory and decision-making functions [15, 16, 17, 18, 19] presents many opportunities in biotechnological applications, a lack of formal understanding associated with genetic context-dependent effects has limited progress in engineering biology. In this respect, two studies have shown that the 5? untranslated region (5?-UTR) of mRNA can affect the temporal control of multigene operons or inverter-based genetic circuits, and RNA processing using clustered regularly interspaced short palindromic repeats (CRISPRs) or ribozymes can serve as effective genetic insulators to buffer such context-dependent effects [5, 20]. In this paper, we have sought to elucidate the limitations of engineering biology from an architectural point of view, with the aim of creating a set of engineering solutions for overcoming failure modes during the development of complex, synthetic genetic circuits.
Design of biological half adder

In this study we were interested in developing biological half adders in prokaryotic systems — particularly in microbes which exhibit much faster cell division and shorter cycle time — so that they can be broadly applied in different biotechnological applications. In contrast to the mammalian cell-based half adder, which is developed mainly for therapeutic and biosensing applications, a prokaryotic half adder can be used to enhance molecular process control and decision making, for example, in drug and biofuel production, biosensing, bioremediation [21] and probiotic engineering for the treatment of metabolic disorders [22], cancer [23] and infectious diseases [24, 25]. In digital processing, half adders form the key building blocks for shift registers, binary counters and serial parallel data converters. Likewise in biological systems, a combination of half adders can be connected in various arrangements to regulate gene expression with diverse, digital-like performance. In doing so, biological systems can be made to interface with novel biomolecular devices, allowing the repurposing of cellular phenotype, as well as providing new platforms to probe and elucidate biological functions