Browsing by Subject "Whole-cell biosensors"

Now showing 1 - 5 of 5

Open Access
Genetic circuits combined with machine learning provides fast responding living sensors
(Elsevier BV, 2021-04-15) Saltepe, Behide; Bozkurt, Eray Ulaş; Güngen, Murat Alp; Çiçek, A. Ercüment; Şeker, Urartu Özgür Şafak
Whole cell biosensors (WCBs) have become prominent in many fields from environmental analysis to biomedical diagnostics thanks to advanced genetic circuit design principles. Despite increasing demand on cost effective and easy-to-use assessment methods, a considerable amount of WCBs retains certain drawbacks such as long response time, low precision and accuracy. Here, we utilized a neural network-based architecture to improve the features of WCBs and engineered a gold sensing WCB which has a long response time (18 h). Two Long-Short Term-Memory (LSTM)-based networks were integrated to assess both ON/OFF and concentration dependent states of the sensor output, respectively. We demonstrated that binary (ON/OFF) network was able to distinguish between ON/OFF states as early as 30 min with 78% accuracy and over 98% in 3 h. Furthermore, when analyzed in analog manner, we demonstrated that network can classify the raw fluorescence data into pre-defined analyte concentration groups with high precision (82%) in 3 h. This approach can be applied to a wide range of WCBs and improve rapidness, simplicity and accuracy which are the main challenges in synthetic biology enabled biosensing.
Open Access
Genetic circuits to detect nanomaterial triggered toxicity through engineered heat shock response mechanism
(American Chemical Society, 2019) Saltepe, Behide; Bozkurt, Eray Ulaş; Hacıosmanoğlu, Nedim; Şeker, Urartu Özgür Şafak
Biocompatibility assessment of nanomaterials has been of great interest due to their potential toxicity. However, conventional biocompatibility tests fall short of providing a fast toxicity report. We developed a whole cell based biosensor to track biocompatibility of nanomaterials with the aim of providing fast feedback to engineer them with lower toxicity levels. We engineered promoters of four heat shock response (HSR) proteins utilizing synthetic biology approaches. As an initial design, a reporter coding gene was cloned downstream of the selected promoter regions. Initial results indicated that native heat shock protein (HSP) promoter regions were not very promising to generate signals with low background signals. Introducing riboregulators to native promoters eliminated unwanted background signals almost entirely. Yet, this approach also led to a decrease in expected sensor signal upon stress treatment. Thus, a repression based genetic circuit, inspired by the HSR mechanism of Mycobacterium tuberculosis, was constructed. These genetic circuits could report the toxicity of quantum dot nanoparticles in 1 h. Our designed nanoparticle toxicity sensors can provide quick reports, which can lower the demand for additional experiments with more complex organisms.
Open Access
Multiplexed cell-based diagnostic devices for the detection of kidney disease pathological markers
(2020-12) Köse, Sıla
Development of accurate, inexpensive and fast-screening devices has been a big vacancy in the field of medical diagnostics. Close monitoring and diagnosis of various diseases are generally conducted by typical analytical techniques which require intensive efforts and qualified personnel. Therefore, there is an urgent need for more convenient and reliable alternatives that are highly specific, cost-efficient and rapid. One of the approaches in solving this problem is using naturally-derived proteins and nucleic acid molecules that can respond to various types of metabolites. Synthetic biology utilizes these components to assemble biological systems that can probe and control metabolic state of a host. In this context, biosensors can be considered highly specific, costeffective and can be implemented in point-of-care bioanalytical tools for effective healthcare. Here, we have developed biosensors that are responsive to medically relevant biomarkers namely; urea and uric acid. Furthermore, a multi-input version that can sense and respond to both biomarkers simultenously and another multiplexed biosensor that can mimic the AND-logic were developed. Biosensors were designed to respond to their respective target analyte presence through an increase in the fluorescence intensity which can be measured with spectrophotometric devices. To do so, native promoter and transcription factors from different organisms were assembled inside a gene circuit to express a fluorescent protein in the presence of the respective biomarker. For urea biosensor, UreR transcriptional activator and the complete intergenic region inside the urease operon of the organism Proteus mirabilis were utilized. The system was optimized to have the desired dose- response curve using post trancriptional regulation elements and protein engineering. For the uric acid biosensor, transcriptional repressor HucR and the transcription factor binding site inside the uricase operon from the organism Deinococcus radiodurans were assembled in a gene circuit as well as a Uric Acid Transporter (UACT). Using promoter engineering and copy number modifications the response curve of the system was optimized. Next, biological components of the biomarkers were assembled in a multiplexed system to respond both molecules simultenously. Furthermore, a logic gate operating system was developed using promoter engineering that performs AND-logic to be implemented in a medically relevant algorithm A framework for stabilization of biosensors on low-cost portable paper discs through biofilmcellulose interactions, and entrapment of whole-cell biosensors inside biocompatible, biodegradable and mechanically strong gelatin beads was provided for remote detection of the pathological biomarkers. Finally, the robustness of the developed whole cell biosensors was tested with human clinical samples.
Open Access
Synthetic genetic circuits for self-actuated cellular nanomaterial fabrication devices
(American Chemical Society, 2019) Ölmez, Tolga Tarkan; Şahin-Kehribar, Ebru; Işılak, Musa Efe; Lu, T. K.; Şeker, Urartu Özgür Şafak
Genetically controlled synthetic biosystems are being developed to create nanoscale materials. These biosystems are modeled on the natural ability of living cells to synthesize materials: many organisms have dedicated proteins that synthesize a wide range of hard tissues and solid materials, such as nanomagnets and biosilica. We designed an autonomous living material synthesizing system consisting of engineered cells with genetic circuits that synthesize nanomaterials. The circuits encode a nanomaterial precursor-sensing module (sensor) coupled with a materials synthesis module. The sensor detects the presence of cadmium, gold, or iron ions, and this detection triggers the synthesis of the related nanomaterial-nucleating extracellular matrix. We demonstrate that when engineered cells sense the availability of a precursor ion, they express the corresponding extracellular matrix to form the nanomaterials. This proof-of-concept study shows that endowing cells with synthetic genetic circuits enables nanomaterial synthesis and has the potential to be extended to the synthesis of a variety of nanomaterials and biomaterials using a green approach.
Open Access
Synthetic genetic circuits to monitor nanomaterial triggered toxicity
(2020-07) Saltepe, Behide
In the past decades, nanomaterial (NM) usage in various fields has been of great interest because of their unique properties that show tuneable optical and physical properties depending on their size. Yet, safety concerns of NMs on human or environment arise with increased NM usage. Thanks to their small size, NMs can easily penetrate through cellular barriers and their high surface-to-volume ratio makes them catalytically active creating stress on cells such as protein unfolding, DNA damage, ROS generation etc. Hence, biocompatibility assessment of NMs has been analyzed before their field application such as drug delivery and imaging which requiring human exposure. Yet, conventional biocompatibility tests fall short of providing a fast toxicity report. One aspect of the present thesis is to develop a living biosensor to report biocompatibility of NMs with the aim of providing fast feedback to engineer them with lower toxicity levels before applying on humans. For this purpose, heat shock response (HSR), which is the general stress indicator, was engineered utilizing synthetic biology approaches. Firstly, four highly expressed heat shock protein (HSP) promoters were selected among HSPs. In each construct, a reporter gene was placed under the control of these HSP promoters to track signal change upon stress (i.e., heat or NMs) exposure. However, initial results indicated that native HSPs are already active in cells to maintain cellular homeostasis. Moreover, they need to be engineered to create a proper stress sensor. Thus, these native HSP promoters were engineered with riboregulators and results indicated that these new designs eliminated unwanted background signals almost entirely. Yet, this approach also led to a decrease in expected sensor signal upon stress treatment. To increase the sensor signal, a positive feedback loop using bacterial communication, quorum sensing, method was constructed. HSR was integrated with QS circuit showed that signal level increased drastically. Yet, background signal also increased. Moreover, instead of using activation based HSR system as in Escherichia coli, repression based system was hypothesized to solve the problem. Thus, a repression based genetic circuit, inspired by the HSR mechanism of Mycobacterium tuberculosis, was constructed. These circuits could report the toxicity of quantum dots (QDs) in 1 hour. As a result, these NM toxicity sensors can provide quick reports, which can lower the demand for additional experiments with more complex organisms. As part of this study, a source detection circuit coupling HSR mechanism with metal induced transcription factors (TFs) has been constructed to report the source of the toxic compound. For this purpose, gold and cadmium were selected as model ions. In the engineered circuits, stress caused by metal ions activates expression of regulatory elements such as TFs of specific ions (GolS for gold and CadR and MerR(mut) for cadmium) and a site-specific recombinase. In the system, the recombinase inverts the promoter induced by TF-metal ion complex, and a reporter has been expressed based on the inducer showing the source of the stress as either gold or cadmium. Finally, a mammalian cellular toxicity sensor has been developed using similar approaches used in bacterial sensors. To begin with, two HSP families have been selected: HSP70 and α-Bcrystallin. Initial circuits were designed using promoter regions of both protein families to control the expression of a reporter, gfp. Both circuits were tested with heat and cadmium ions with varying concentrations and results showed that HSP70-based sensor had high background signal because of its active role in cellular homeostasis and protein folding in cells. Additionally, a slight increase was observed after heat treatment. Similar results were observed for α-Bcrystallin-based sensor; yet, these outcomes were not suitable for a desirable sensor requiring tight control. Thus, we decided to transfer the bacterial repression based toxicity sensor into mammalian cells. At the beginning, expression of the repressor, HspR, from M. tuberculosis was checked in HEK293T cell line and modified with nuclear localization signal (NLS) to localize the repressor in the nucleus. Further, a minimal promoter (SV40) controlling the expression of a reporter was engineered with single and double inverted repeats (IRs) for HspR binding. Then, HspR and engineered reporter circuits were co-trasfected to track signals at normal growth conditions and upon stress. Each circuit was tested with heat and cadmium treatment and results were showed repression of GFP expression by HspR at normal conditions, but no significant signal increase was observed upon stress. Hence, constructed mammalian circuits require more optimization to find optimum working conditions of sensors. To sum up, in this study, a powerful candidate to manufacture ordered gene circuits to detect nanomaterial-triggered toxicity has been demonstrated. Unlike previous studies utilizing HSR mechanism as stress biosensors, we re-purposed the HSR mechanism of both bacteria and mammalian cells with different engineering approaches (i.e., riboregulators, quorum sensing mechanism, promoter engineering). As a result, an easy-to-use, cheap and fast acting nanomaterial-triggered toxicity assessment tool has been developed. Also, initial principles of mammalian whole cell biosensor design for the same purpose have been indicated to expand the limited toxicity detection strategies utilizing mammalian cells. This study contributed for the detection of toxic NMs providing a feedback about the fate of these NMs so that one can engineer them to make biocompatible before field application.