Intein-mediated thyroid hormone biosensors: towards controlled delivery of hormone therapy

Abstract

Although blood sampling and medical imaging are well-established techniques in clinical diagnostics, they often require long post-processing procedures. Fast and simple quantification of signaling molecules can enable efficient health monitoring and improve diagnoses. Thyroid hormones (THs) treatment relies on trial-and-error dose adjustments, and requires constant tracking via blood tests. Thus, a fast and reliable method that can constantly track THs levels could substantially improve patient quality of life by reducing their visits to doctors. Synthetic biosensors have shown to be inexpensive and easy tools for sensing molecules, with their use in healthcare increasing over time. This study describes the construction of an engineered THs bacterial biosensor, consisting of a split-intein-based TH receptor ligand binding domain (LBD) biosensor that reconstitutes green fluorescence protein (GFP) after binding to TH. This biosensor could quantitatively measure THs concentrations by evaluating fluorescence intensity. In vitro sensing using Escherichia coli produced GFP over a wide dynamic range. The biosensor was further optimized by adding a double LBD, which enhanced its dynamic range until it reached healthy physiological conditions. Moreover, a mathematical model was developed to assess the dynamic properties of the biosensor and to provide a basis for the characterization of other intein-mediated biosensors. This type of biosensor can be used as the basis for novel treatments of thyroid diseases and can be adapted to measure the concentrations of other hormones, giving rise to a series of mathematically characterized modular biosensors.

Keywords: biosensor; hypothyroidism; intein; mathematical modeling; synthetic biology; thyroid hormones.

FIGURE 1

THs sensing at the body (A) and molecular (B, C) levels. (A) The hypothalamic-pituitary-thyroid axis. When the hypothalamus senses low levels of THs, it releases TRH, which activates the pituitary gland to release TSH. TSH, in turn, stimulates the thyroid gland to produce THs that are released to the systemic circulation. When the hypothalamus senses that levels of TH are sufficient, it stops releasing TRH, so that no more TH are produced (Dietrich et al., 2012). (B) THR activation molecular mechanism. THRs are a group of nuclear proteins that can bind to specific hormones through their LBDs, which interact with T3 (Yen, 2001). This is followed by the binding of the THR to a specific sequence using its DBD, and the recruiting of proteins to unpack the chromatin and induce gene transcription (Flamant et al., 2017). (C) Human THR- β ₁ isoform. The human THR-β ₁ contains a hinge region between the LBD and DBD that provides the receptor with enough flexibility to change between its active and inactive forms. The LBD, DBD and hinge region are embedded by the activation functions (AF) AF1 and AF2 at the N- and C-termini, respectively. These AFs allow the THR-β ₁ receptor to bind to coactivators to initiate transcriptional activation of target genes.

FIGURE 2

Schematic representation of sfIMT3 activity upon binding T3. sfIMT3 consists of the THR-β ₁ LBD fused with the splicing domains of the Mtu RecA intein, with spliced sfGFP acting as a reporter (see Section 2.1 for more information). The binding of T3 to the LBD results in the reconstruction of the sfGFP by the splicing domains of the inteins. This system creates a Hill shaped transfer function that connects the T3 concentration in the media to the fluorescence emitted by the sensor cells. In addition, a geometrical definition of the amplitude of a general biosensor (γ) and its dynamic range is shown. The boundaries are indicated by the points at which the green tangential line intersects with the maximum and minimum output values of the biosensor (see Eqs 7–9).

FIGURE 3

Western blotting of BL21 expressing eIMT3 using a SDS-PAGE gel. Cells were grown in fresh LB media + Kanamycin 25 μg/mL with 10^–4 M T3 (+) or in the absence of T3 (−) overnight until reaching saturation (Western blot protocol can be found in Section 2.2). Controls show the presence of the entire construct (85 kDa), whereas cells treated with T3 show higher concentrations of the eGFP after splicing (37 kDa).

FIGURE 4

Transfer functions correlating the emitted fluorescence with the amount of thyroid hormone of the biosensors (A) sfIMT3 induced with T3 (B) sfIMT3 induced with T4 (C) dIMT3 induced with T3 (D) dIMT3 induced with T4. Cells were grown in fresh LB media + Carbenicillin 25 μg/mL overnight until they attained an OD ₆₀₀ of 0.4–0.6. A 100 μL aliquot of cultured cells was added to each well of a 96 well black/clear bottom plate, along with 100 μL of the 2x ligand solutions. Experimental data represents the mean value of six replicates per TH concentration at 30 h after TH exposure, with error bars showing its standard error. The transfer functions were fitted using the least-squares method.