Engineered bacteria (synthetic biotics) represent a new class of therapeutics that leverage the tools of synthetic biology. Translational testing strategies are required to predict synthetic biotic function in the human body. Gut-on-a-chip microfluidics technology presents an opportunity to characterize strain function within a simulated human gastrointestinal tract. Here, we apply a human gut-chip model and a synthetic biotic designed for the treatment of phenylketonuria to demonstrate dose-dependent production of a strain-specific biomarker, to describe human tissue responses to the engineered strain, and to show reduced blood phenylalanine accumulation after administration of the engineered strain. Lastly, we show how in vitro gut-chip models can be used to construct mechanistic models of strain activity and recapitulate the behavior of the engineered strain in a non-human primate model. These data demonstrate that gut-chip models, together with mechanistic models, provide a framework to predict the function of candidate strains in vivo.

The DNA sequence encoding the rbs repressor protein, RbsR, has been determined. Amino acid sequence analyses of the product of an rbsR-lac2 fusion and of affinity-purified RbsR demonstrate that translation begins at an unusual codon, TTG, and that the initial amino acid is removed during maturation of the protein. DNA-binding assays indicate that RbsR binds to a region of perfect dyad symmetry spanning the rbs operon transcriptional start site and that the affinity for the rbs operator is reduced by addition of ribose, consistent with ribose being the inducer of the operon. RbsR is a member of a family of homologous repressor proteins having very similar DNA-binding sites and binding to similar operator sequences. [RbsR PIR accession number A41828.1 Keywords: Escherichia coli; repressors; ribose transport and utilization; transcription regulationThe rbs operon of Escherichia coli K12 encodes the highaffinity membrane transport system for ribose (Iida et al., 1984; Lopilato et al., 1984). Three open reading frames at the 5' end of the mRNA, designated rbsD, rbsA, and rbsC , are located in the region shown to be necessary for the membrane transport function (Iida et al., 1984;Lopilato et al., 1984). The coding region for the periplasmic ribose binding protein, rbsB, follows (Groarke et al., 1983), with that for ribokinase, rbsK , completing the set of genes that have been demonstrated to function as a single transcriptional unit (Lopilato et al., 1984).The gene encoding the repressor for the rbs operon, rbsR, was shown to be located downstream from rbsK, and evidence indicated that it is on a separate transcriptional unit from rbs (Lopilato et al ., 1984). During the sequence analysis, we found an open reading frame immediately downstream of rbsK that was clearly homologous to lacland galR . We have now completed the sequence analysis of this open reading frame and have shown that it encodes RbsR, a member of a family of repressor proteins. Functional studies have confirmed that the protein is expressed and binds to DNA sequences upstream of rbsD that conform to the consensus operator sequences for this family of repressors. These studies, together with the earlier genetic analyses (Lopilato et al., 1984), identify and locate the rbsR gene.

The deduced amino acid sequence of the rbs repressor, RbsR, of Escherichia coli is homologous over its C-terminal 272 residues to the entire sequence of the periplasmic ribose binding protein. RbsR is also homologous to a family of bacterial repressor proteins including LacI. This implies that the structure of the repressor consists of a two-domain binding protein portion attached to a DNA-binding domain having the four-helix structure of the Lac1 headpiece. The implications of these relationships to the mechanism of this class of repressors are discussed.

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Synbiotics are a new class of live therapeutics employing engineered genetic circuits. The rapid adoption of genetic editing tools has catalyzed the expansion of possible synbiotics, exceeding traditional testing paradigms in terms of both throughput and model complexity. Herein, we present a simplistic gut-chip model using common Caco2 and HT-29 cell lines to establish a dynamic human screening platform for a cortisol sensing tryptamine producing synbiotic for cognitive performance sustainment. The synbiotic, SYN, was engineered from the common probiotic E. coli Nissle 1917 strain. It had the ability to sense cortisol at physiological concentrations, resulting in the activation of a genetic circuit that produces tryptophan decarboxylase and converts bioavailable tryptophan to tryptamine. SYN was successfully cultivated within the gut-chip showing log-phase growth comparable to the wild-type strain. Tryptophan metabolism occurred quickly in the gut compartment when exposed to 5 μM cortisol, resulting in the complete conversion of bioavailable tryptophan into tryptamine. The flux of tryptophan and tryptamine from the gut to the vascular compartment of the chip was delayed by 12 h, as indicated by the detectable tryptamine in the vascular compartment. The gut-chip provided a stable environment to characterize the sensitivity of the cortisol sensor and dynamic range by altering cortisol and tryptophan dosimetry. Collectively, the human gut-chip provided human relevant apparent permeability to assess tryptophan and tryptamine metabolism, production, and transport, enabled host analyses of cellular viability and pro-inflammatory cytokine secretion, and succeeded in providing an efficacy test of a novel synbiotic. Organ-on-a-chip technology holds promise in aiding traditional therapeutic pipelines to more rapidly down select high potential compounds that reduce the failure rate and accelerate the opportunity for clinical intervention.