Bacterial defiance as a form of prodrug failure

Brandon Alexander Holt; Gabriel A. Kwong

Under this analogy, we postulated that multiple prodrug biological transistors could be used to construct logical operations (i.e., AND, OR, NOT gates) that allow for the design of integrated biological circuits that output state "0" (i.e., bacterial death) for all possible inputs. These biological 15 circuits would then be representative of a multi-prodrug strategy to eliminate bacteria even when input BAH variables (i.e., temperature, nutrient level, enzyme activity) would result in a state of defiance for a single prodrug. To accomplish this, we designed complementary N-type and P-type transistors, which are required to construct all possible logic gates 40 . N-type transistors allow input current to pass when the gate signal is above a defined threshold, whereas P-type transistors 20 allow current to pass when the gate signal is below a defined threshold. We constructed N-type transistors with our AMP prodrug system, which allowed input bacteria (Bin = 1) to survive (Bout = 1) when the BAH is above BAHcrit which we used to define the gate threshold (i.e., BAH > BAHcrit equivalent to G = 1; BAH < BAHcrit equivalent to G = 0) (Fig. 4B) . To create P-type transistors, we synthesized heat-triggered liposomes 41 (Fig. S4) loaded with the antibiotic ampicillin 42 , which was 25 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/556951 doi: bioRxiv preprint released at temperatures above 37 o C to kill bacteria (i.e., Bout = 0) (Fig. 4C) . Above this critical temperature, treatment with AMP prodrugs leads to bacterial defiance (i.e., BAH > BAHcrit or G = 1) and E. coli survive prodrug treatment by proliferating significantly faster than prodrug activation 43 . We therefore defined temperatures above 37 o C as corresponding to G = 1 and below as G = 0 for our P-type biological transistor to match inverse gate values for our N-type biological 5