Bacterial Hash Function Using DNA-Based XOR Logic Reveals Unexpected Behavior of the LuxR Promoter

Introduction: Hash functions are computer algorithms that protect information and secure transactions. In response to the NIST’s "International Call for Hash Function”, we developed a biological hash function using the computing capabilities of bacteria. We designed a DNA-based XOR logic gate that allows bacterial colonies arranged in a series on an agar plate to perform hash function calculations. Results and Discussion: In order to provide each colony with adequate time to process inputs and perform XOR logic, we designed and successfully demonstrated a system for time-delayed bacterial growth. Our system is based on the diffusion of -lactamase, resulting in destruction of ampicillin. Our DNA-based XOR logic gate design is based on the opposition of two promoters. Our results showed that Plux and POmpC functioned as expected individually, but Plux did not behave as expected in the XOR construct. Our data showed that, contrary to literature reports, the Plux promoter is bidirectional. In the absence of the 3OC6 inducer, the LuxR activator can bind to the Plux promoter and induce backwards transcription. Conclusion and Prospects: Our system of time delayed bacterial growth allows for the successive processing of a bacterial hash function, and is expected to have utility in other synthetic biology applications. While testing our DNA-based XOR logic gate, we uncovered a novel function of Plux. In the absence of autoinducer 3OC6, LuxR binds to Plux and activates backwards transcription. This result advances basic research and has important implications for the widespread use of the Plux promoter.