Easy-to-perform, relatively inexpensive blood diagnostics have transformed at-home healthcare for some patients, but they require analytical equipment and are not easily adapted to measuring other biomarkers. The requirement for reliable quantification in complex sample types (such as blood) has been a critical roadblock in developing and deploying inexpensive, minimal-equipment diagnostics. Here, we developed a platform for inexpensive, easy-to-use diagnostics that uses cell-free expression to generate colored readouts that are visible to the naked eye, yet quantitative and robust to the interference effects seen in complex samples. We achieved this via a parallelized calibration scheme that uses the patient sample to generate custom reference curves. We used this approach to quantify a clinically relevant micronutrient and to quantify nucleic acids, demonstrating a generalizable platform for low-cost quantitative diagnostics.

Biotechnology has transformed the production of various chemicals and pharmaceuticals due to its efficient and selective processes, but it is inherently limited by its use of live cells as "biocatalysts."Cell-free expression (CFE) systems, which use a protein lysate isolated from whole cells, have the potential to overcome these challenges and broaden the scope of biomanufacturing. Implementation of CFE systems at scale will require determining clear markers of lysate activity and developing supplementation approaches that compensate for potential variability across batches and experimental protocols. Toward this goal, we use metabolomics to relate lysate preparation and performance to metabolic activity. We show that lysate processing affects the metabolite makeup of lysates and that lysate metabolite levels change over the course of a CFE reaction regardless of whether a target compound is produced. Finally, we use this information to develop ways to standardize lysate activity and to design an improved CFE system.

Pigmented metabolites have great potential for use in biosensors that target low-resource areas, since sensor output can be interpreted without any equipment. However, full repression of pigment production when undesired is challenging, as even small amounts of enzyme can catalyze the production of large, visible amounts of pigment. The red pigment lycopene could be particularly useful because of its position in the multi-pigment carotenoid pathway, but commonly used inducible promoter systems cannot repress lycopene production. In this paper, we designed a system that could fully repress lycopene production in the absence of an inducer and produce visible lycopene within two hours of induction. We engineered Lac, Ara, and T7 systems to be up to 10 times more repressible, but these improved systems could still not fully repress lycopene. Translational modifications proved much more effective in controlling lycopene. By decreasing the strength of the ribosomal binding sites on the crtEBI genes, we enabled full repression of lycopene and production of visible lycopene in 3–4 hours of induction. Finally, we added the mevalonate pathway enzymes to increase the rate of lycopene production upon induction and demonstrated that supplementation of metabolic precursors could decrease the time to coloration to about 1.5 hours. In total, this represents over an order of magnitude reduction in response time compared to the previously reported strategy. The approaches used here demonstrate the disconnect between fluorescent and metabolite reporters, help enable the use of lycopene as a reporter, and are likely generalizable to other systems that require precise control of metabolite production.

Synthetic biology seeks to redesign biological systems to perform novel functions in a predictable manner. Recent advances in bacterial and mammalian cell engineering include the development of cells that function in biological samples or within the body as minimally invasive diagnostics or theranostics for the real-time regulation of complex diseased states. Ex vivo and in vivo cell-based biosensors and therapeutics have been developed to target a wide range of diseases including cancer, microbiome dysbiosis and autoimmune and metabolic diseases. While probiotic therapies have advanced to clinical trials, chimeric antigen receptor (CAR) T cell therapies have received regulatory approval, exemplifying the clinical potential of cellular therapies. This Review discusses preclinical and clinical applications of bacterial and mammalian sensing and drug delivery platforms as well as the underlying biological designs that could enable new classes of cell diagnostics and therapeutics. Additionally, we describe challenges that must be overcome for more rapid and safer clinical use of engineered systems.

Micronutrient deficiencies, including zinc deficiency, are responsible for hundreds of thousands of deaths annually. A key obstacle to allocating scarce treatment resources is the ability to measure population blood micronutrient status inexpensively and quickly enough to identify those who most need treatment. This paper develops a metabolically engineered strain of Escherichia coli to produce different colored pigments (violacein, lycopene, and β-carotene) in response to different extracellular zinc levels, for eventual use in an inexpensive blood zinc diagnostic test. However, obtaining discrete color states in the carotenoid pathway required precise engineering of metabolism to prevent reaction at low zinc concentrations but allow complete reaction at higher concentrations, and all under the constraints of natural regulator limitations. Hence, the metabolic engineering challenge was not to improve titer, but to enable precise control of pathway state. A combination of gene dosage, post-transcriptional, and post-translational regulation was necessary to allow visible color change over physiologically relevant ranges representing a small fraction of the regulator’s dynamic response range, with further tuning possible by modulation of precursor availability. As metabolic engineering expands its applications and develops more complex systems, tight control of system components will likely become increasingly necessary, and the approach presented here can be generalized to other natural sensing systems for precise control of pathway state.

Metabolic engineering is generally focused on static optimization of cells to maximize production of a desired product, though recently dynamic metabolic engineering has explored how metabolic programs can be varied over time to improve titer. However, these are not the only types of applications where metabolic engineering could make a significant impact. Here, we discuss a new conceptual framework, termed “precision metabolic engineering,” involving the design and engineering of systems that make different products in response to different signals. Rather than focusing on maximizing titer, these types of applications typically have three hallmarks: sensing signals that determine the desired metabolic target, completely directing metabolic flux in response to those signals, and producing sharp responses at specific signal thresholds. In this review, we will first discuss and provide examples of precision metabolic engineering. We will then discuss each of these hallmarks and identify which existing metabolic engineering methods can be applied to accomplish those tasks, as well as some of their shortcomings. Ultimately, precise control of metabolic systems has the potential to enable a host of new metabolic engineering and synthetic biology applications for any problem where flexibility of response to an external signal could be useful.

Bacterial biosensors can enable programmable, selective chemical production, but difficulties incorporating metabolic pathways into complex sensor circuits have limited their development and applications. Here we overcome these challenges and present the development of fast-responding, tunable sensor cells that produce different pigmented metabolites based on extracellular concentrations of zinc (a critical micronutrient). We create a library of dual-input synthetic promoters that decouple cell growth from zinc-specific metabolite production, enabling visible cell coloration within 4 h. Using additional transcriptional and metabolic control methods, we shift the response thresholds by an order of magnitude to measure clinically relevant zinc concentrations. The resulting sensor cells report zinc concentrations in individual donor serum samples; we demonstrate that they can provide results in a minimal-equipment fashion, serving as the basis for a field-deployable assay for zinc deficiency. The presented advances are likely generalizable to the creation of other types of sensors and diagnostics.

Cell-free systems provide a versatile platform for the development of low-cost, easy-to-use sensors for diverse analytes. However, sensor affinity dictates response sensitivity, and improving binding affinity can be challenging. Here, we describe efforts to address this problem while developing a biosensor for vitamin B12, a critical micronutrient. We first use a B12-responsive transcription factor to enable B12-dependent output in a cell-free reaction, but the resulting sensor responds to B12 far above clinically relevant concentrations. Surprisingly, when expressed in cells, the same sensor mediates a much more sensitive response to B12. The sensitivity difference is partly due to regulated import that accumulates cytoplasmic B12. Overexpression of importers further improves sensitivity, demonstrating an inherent advantage of whole-cell sensors. The resulting cells can respond to B12 in serum, can be lyophilized, and are functional in a minimal-equipment environment, showing the potential utility of whole-cell sensors as sensitive, field-deployable diagnostics.