Difference between revisions of "Team:NTHU Formosa/Design"

Design

Autonomous bioluminescence output in mammalian cell system induced by extracellular soluble stimuli

Claiming the versatility of such programmable signaling pathways inducible by extracellular soluble molecules following autonomous regulation of the downstream gene expression, we propose a customer-oriented application in diagnostic aspect with considerately high commercial potential. In the extracellular space such as bloodstream, various soluble proteins serve as biomarkers representing the physiological or pathological conditions of organisms (Altintas and Tothill, 2013; Rapisuwon et al., 2016). For the purpose of diagnosis, blood test is one of the most common way for detecting biomarkers but it suffers from several inevitable drawbacks such as invasiveness, time-consuming procedure, demand for medical staff service, non-real-time tracking and so on. These disadvantages may discourage people from the periodic medical checkup. In terms of non-invasive real-time health monitoring, great varieties of wearable devices and smartwatches, working as personal daily fitness trackers, have been successively developed. The global market of wearable medical devices is estimated to reach $35 billion USD soon. However, most of the current devices are only capable of providing limited diagnosis or information, such as heart rate, steps taken, calories, quality of sleep and a few other personal metrics, which cannot precisely represent the physiological states relating to innumerable life-threatening diseases.

　 To our knowledge, this is the first programmable system for non-invasive real-time diagnosis capable of receiving the soluble biomarkers as input stimulation from extracellular space and triggering the autonomous bioluminescence output. Powerful and versatile, our design is anticipated to revolutionize the strategies of non-invasive real-time diagnoses.

　 To address this long-standing worldwide issue, we developed engineered cells that are capable of receiving extracellular soluble biomarkers as stimuli and autonomously produce bioluminescence signal output for real-time and non-invasively diagnosis purpose based on our programmable signaling pathway. Conventionally, the production of bioluminescence requires additional chemical substrates, luciferins for example, which undergo an enzyme-catalysed oxidation resulting in emitting bioluminescence signal (Mezzanotte et al., 2017). Injecting luciferins into living host is the invasive procedures we struggled to avoid. Therefore, for the output signal, we adopted a codon-optimized autonomous bioluminescence system, lux gene cassette, originated from microbes. This codon-optimized lux bioluminescence system scavenges endogenous intermediated metabolites stock within mammalian cells and converts them into substrates by enzymes, hLuxC, hLuxD, hLuxE, and frp (Close et al., 2010; Xu et al., 2014). To this end, CMV promoter is designated to continuously drive the expression of hLuxC, hLuxD, hLuxE, and frp for substrates production, while TRE3G promoter is used to inducibly drive the expression of hLuxA and hLuxB to form luciferase dimer (Fig. 6). Bioluminescent output can be obtained in the presence of both substrates and inducibly formed luciferase when both promoters are activated.

Figure 1. Schematic representation of our programmable signaling pathways.

　 For the details of our bio-mechanism, mesenchymal stem cells (MSCs ^[1]) will play the role as the reporter cell, carrying the gene circuit we constructed. Nanobodies ^[2], expressed extracellularly, act as the detectors for their ability of binding specific bio-factors. To further enhance the accuracy of nanobodies binding to the correct target, we apply notch system here. Basically, nanobodies are separated into two terminals, and only the binding of biomarkers stimulates the combination of two nanobody segments. As the split nanobodies segments approached each other, the split TEV protease segments conjugated to split-GBP are brought together and become functional. The functional TEV protease recognize and cleave the TEV cutting site, followed by the translocation of tTA transcription factor into the nucleus, where tTA triggers the production of luciferase, LuxAB and complete the luciferase pathway ^[3] (Lux gene metabolic pathway). Thus, bioluminescence is generated at the end. Based on previous researches and experimental experiences, this wavelength range of light is too weak to be visible but still capable of penetrating through blood vessels then skin. We assume that in the upcoming future, the PMT detector recording bioluminescence signal is adapted for installation on a watch. As the “glowing” cells pass by your wrist, the watch could then document the present health condition. In addition, our team would program applications to analyze all the historical data for more convenient and readable user view. To begin with, checking whether our construct works, we chose HEK293T as the reporter cells, and replace a segment of detector from nanobody to GFP-binding protein (GBP), with the usage of Green fluorescent protein (GFP) as the substrate (Fig.2).

Figure 2. Biowatcher Reporter cells

1. Mesenchymal stem cells (MSCs):
Connective tissue cells which are multipotent and able to perform cell renewal.

2. Nanobodies:
Single variable domain derived from antibody fragment, with the size of 3nm (15kDa). Their small size enables them to pass through body tissues faster. Also, the simple structure results in higher affinity to antigen in contrast to the complete antibody.

3. Luciferase:
Enzyme that catalyzes the pathway of emitting light, with the existence of substrates. Bioluminescence, namely the emitted light, is at its maximum intensity at 490nm, which is close to blue-green colored