Synthetic biology devices for in vitro and in vivo diagnostics

Abstract

There is a growing need to enhance our capabilities in medical and environmental diagnostics. Synthetic biologists have begun to focus their biomolecular engineering approaches toward this goal, offering promising results that could lead to the development of new classes of inexpensive, rapidly deployable diagnostics. Many conventional diagnostics rely on antibody-based platforms that, although exquisitely sensitive, are slow and costly to generate and cannot readily confront rapidly emerging pathogens or be applied to orphan diseases. Synthetic biology, with its rational and short design-to-production cycles, has the potential to overcome many of these limitations. Synthetic biology devices, such as engineered gene circuits, bring new capabilities to molecular diagnostics, expanding the molecular detection palette, creating dynamic sensors, and untethering reactions from laboratory equipment. The field is also beginning to move toward in vivo diagnostics, which could provide near real-time surveillance of multiple pathological conditions. Here, we describe current efforts in synthetic biology, focusing on the translation of promising technologies into pragmatic diagnostic tools and platforms.

Keywords: biosensing; diagnostics; nanobiotechnology; synthetic biology; synthetic gene networks.

Fig. 1.

RNA-based biosensors and synthetic biology platforms for in vitro diagnostics. (A) Conventional riboregulators inhibit the translation of mRNA from the start codon (AUG) by sequestering the RBS through a cis-repression sequence (Cis), which is relieved in the presence of the transactivator RNA (taRNA). (B) In the toehold switch model, the RBS is located in a hairpin loop within the repressed RNA’s 5′ untranslated region and a toehold is added to the 5′ end. This alternative regulatory RNA structure allows for a much larger RNA sequence space to be detected. (C) In vitro phage-based diagnostics rely on specific recognition of target bacterial species by engineered phage particles. Once the phage has bound, the engineered phage genome is injected into the targeted cells, where the reporter gene is expressed (i.e., luciferase; yellow circles) and phage replicate. (D) Paper-based systems are assembled by freeze-drying a diagnostic gene network and a cell-free coupled transcription/translation system into paper or other porous materials. The gene circuit becomes active when rehydrated with the test sample, containing target RNAs or small molecules.

Fig. 2.

Synthetic biology devices for in vivo diagnostics. (A) The CaspaseTracker: Cytoplasmic Gal4 (blue) is released to the nucleus by apoptotic caspases (orange), where it activates RFP and FLP recombinase (violet star) expression, leading to persistent GFP expression and enabling apoptosis/anastasis tracking (demonstrated in fruit flies). (B) Engineered bacteria naturally home in on tumors and express their synthetic circuits, producing bioluminescence for in situ tumor imaging. (C) The cell-type “classifier” compares endogenous expression levels of three “high” miRNAs (left) and three “low” miRNAs (right) to a preset HeLa profile. If all of the high miRNAs are above the threshold, this profile leads to RNAi silencing of the transactivator (rtTA; blue) of the output gene’s repressor (LacI; orange), allowing for output expression (DsRed; red dots). If all of the low miRNAs are below the threshold, the output mRNA is not degraded via RNAi (demonstrated in cell culture). (D) High urate levels lead to tumor lysis syndrome and gout. A prosthetic urate homeostasis system transports urate (red cogs) into the encapsulated cells via constitutive URAT1 (orange) expression, where it releases mUTS’s (blue) repression of smUox (violet), a secreted uricase that converts the urate to renally secretable allantoin (red dots). (E) Bacteria engineered to record mammalian gut microbiome exposure events. The memory element (M), comprised of a bacteriophage lambda cI/cro (blue/orange) switch and containing a lacZ reporter (violet), is toggled to the “on” (cro, lacZ) state when the Trigger element (T) detects ATc (black cogs) and TetR (green) repression of a second cro copy is released. The detection of ATc results in the expression of β-gal from the lacZ gene, which is readily measured in fecal samples.