Synthetic Gene Circuits Enable Systems-Level Biosensor Trigger Discovery at the Host-Microbe Interface

Abstract

Engineering synthetic circuits into intestinal bacteria to sense, record, and respond to in vivo signals is a promising new approach for the diagnosis, treatment, and prevention of disease. However, because the design of disease-responsive circuits is limited by a relatively small pool of known biosensors, there is a need for expanding the capacity of engineered bacteria to sense and respond to the host environment. Here, we apply a robust genetic memory circuit in Escherichia coli to identify new bacterial biosensor triggers responding in the healthy and diseased mammalian gut, which may be used to construct diagnostic or therapeutic circuits. We developed a pipeline for rapid systems-level library construction and screening, using next-generation sequencing and computational analysis, which demonstrates remarkably reliable identification of responsive biosensor triggers from pooled libraries. By testing libraries of potential triggers—each consisting of a promoter and ribosome binding site (RBS)—and using RBS variation to augment the range of trigger sensitivity, we identify and validate triggers that selectively activate our synthetic memory circuit during transit through the gut. We further identify biosensor triggers with increased response in the inflamed gut through comparative screening of one of our libraries in healthy mice and those with intestinal inflammation. Our results demonstrate the power of systems-level screening for the identification of novel biosensor triggers in the gut and provide a platform for disease-specific screening that is capable of contributing to both the understanding and clinical management of intestinal illness. IMPORTANCE The gut is a largely obscure and inaccessible environment. The use of live, engineered probiotics to detect and respond to disease signals in vivo represents a new frontier in the management of gut diseases. Engineered probiotics have also shown promise as a novel mechanism for drug delivery. However, the design and construction of effective strains that respond to the in vivo environment is hindered by our limited understanding of bacterial behavior in the gut. Our work expands the pool of environmentally responsive synthetic circuits for the healthy and diseased gut, providing insight into host-microbe interactions and enabling future development of increasingly complex biosensors. This method also provides a framework for rapid prototyping of engineered systems and for application across bacterial strains and disease models, representing a practical step toward the construction of clinically useful synthetic tools.

Synthetic Cassettes for pH-Mediated Sensing, Counting, and Containment

“…We next constructed a counter circuit to allow our sensors to record multiple discrete pulses of a given stimulus. We built upon our previous design of a bacterial memory circuit, which is based on the O R operon of bacteriophage l and is capable of sensing and recording transient stimuli (Kotula et al, 2014;Naydich et al, 2019;Riglar et al, 2017). We created a 2-counter circuit ( Figure 3A), based on the O R operon of bacteriophage 434, which produces a fluorescent response only after sensing 2 distinct pulses of a stimulus (e.g., low pH).…”

Synthetic cassettes for pH-mediated sensing, counting and containment

“…We next constructed a counter circuit to allow our sensors to record multiple discrete pulses of a given stimulus. We built upon our previous design of a bacterial memory circuit, which is based on the OR operon of bacteriophage λ, and which is capable of sensing and recording transient stimuli (Kotula et al, 2014;Naydich et al, 2019;Riglar et al, 2017). We created a two-counter circuit ( Figure 3A), based on the OR operon of bacteriophage 434, which produces a fluorescent response only after sensing two distinct pulses of a stimulus (e.g., low pH).…”

“…We constructed a two-counter memory circuit (tc-Ptet-memory), which allows both sensing and recording of two discrete stimulus pulses ( Figure 3D). The tc-Ptet-memory consists of a trigger, an actuator based on the phage 434 cI-Cro switch, and a memory switch based on the phage λ cI-Cro switch (Kotula et al, 2014;Naydich et al, 2019;Riglar et al, 2017). With the first aTc application, the circuit behaves identically to the 434 two-counter, producing Xis and Int from the actuator with the falling edge of the first pulse.…”

“…With the first aTc application, the circuit behaves identically to the 434 two-counter, producing Xis and Int from the actuator with the falling edge of the first pulse. On the second aTc application, the trigger produces λ cIDN (Naydich et al, 2019), which flips the λ memory switch to the Cro (memory-on) state so that it continuously produces a LacZ reporter. As with the previous twocounter, the two-counter memory circuit is designed to respond to the falling edge of the first stimulus pulse and the rising edge of the second.…”

“…From these second group of plasmids, triggers were subsequently integrated into the K-12 MG1655 strain containing the appropriate actuator using the Tn7 transposon. For strain the tc-memory strains: The 434 actuator element was integrated into a TB10 strain via λ Red and subsequently P1 transduced into an E. coli NGF-1 strain that contains a λ memory element (Naydich et al, 2019). The trigger element was then integrated into this strain using the Tn7 transposon as described above.…”