Towards a Complete Set of Genetic Arithmetic Operators: Implementing a Genetic Operational Amplifier Using RNA Regulators

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Yen-Hsiang Wang, Christina Smolke

Stanford University, United States

Biological systems maintain proper functioning in changing environments by performing complex computations on environmental inputs and determining appropriate responses. These biological computations can be constructed in a bottom-up approach by fundamental additive and subtractive operators. Despite a growing number of examples of synthetic biological devices that execute computational functions, limited effort has been directed toward building subtractive devices. We have designed a genetic operational amplifier (gen-opamp), which serves as a key subtractive component in most circuit systems. This gen-opamp consists of three stages: a differential sensor, an amplifier, and an output stage. The differential sensor contains a molecular transducer, which maps the input levels to mRNA levels, and a RNA sense-antisense subtractor as the core of the differential calculation. The genetic differential signal is amplified by a transcriptional device, which is subsequently used to regulate the expression of protein outputs to reveal the comparison result. The non-feedback configuration of the gen-opamp can function as a comparator, whereas the feedback level-setter can keep the ratio of the input levels by actively re-adjusting one to the other. We implemented this gen-opamp using theophylline and tetracycline as the inputs. The corresponding aptamer-coupled ribozymes are implemented as molecular transducers within the targeted mRNAs, together with a mutual complementary 150-bp sense-antisense pair to achieve subtraction. The amplifier is implemented using a synthetic LexA-based promoter regulating either an activator (caffeine demethylase) or deactivator (theophylline demethylase) to form an enzymatic feedback loop targeting the theophylline input. Preliminary results suggest that each of stages of the gen-opamp exhibit desired activity when characterized individually; further partial assembly of each stage proves their compatibility and functionality as a whole. Building a gen-opamp can be a significant milestone towards designing more sophisticated genetic circuits in synthetic biology and understanding the nature of differential genetic interactions in biological networks.