Theses supervised by Dr. Öğr. Üyesi Urartu Özgür Şafak Şeker
14 theses · İhsan Doğramacı Bilkent University
Kırım-Kongo kanamalı ateşine (KKKA) karşı tedavi stratejilerinin geliştirilmesi
Crimean-Congo Hemorrhagic Fever (CCHF) is a highly fatal and zoonotic arboviral infection in humans. It is caused by the CCHF virus, which precipitates severe hemorrhagic outbreaks with a mortality rate reaching up to 40%. CCHF is classified as an arboviral disease due to its transmission through tick vectors of the Hyalomma species, which are arthropods. Human infection occurs either via bites from infected ticks or through exposure to the bodily fluids of infected animals or patients. Since the initial reported case in Turkey in 2002, a total of 9,700 cases have been documented. The disease is endemic in Turkey, positioning it as one of the most affected countries by CCHF within the European region. Currently, there is no effective protective measure, such as a vaccine or specific antiviral treatment, for CCHF. This lack of effective countermeasures constitutes a significant public health threat and a serious sociological issue. The aim of this study is to develop virus-specific and host-safe strategies against CCHF to directly inhibit virus infection and/or subsequent treatment of the disease. For this purpose, the CRISPR/Cas13b genome editing tool will be utilized. RNA editing systems, such as CRISPR-Cas13b, offer the advantage of controlling gene expression without altering the underlying DNA sequence and can exert transient modifications at the RNA level, making them promising tools in combating RNA viruses. The two virus genome segments, the S segment, which encodes the nucleocapsid protein to protect and organize the viral genome, and the L segment, which encodes RNA polymerase for replication and transcription processes necessary for viral propagation, will be rendered ineffective using the CRISPR/Cas tool. Finally, to demonstrate the inhibition of CCHFV replication in HEK293 cells via the CRISPR/Cas13b system, recombinant adeno-associated virus (rAAV) will be used as the gene delivery agent. If the proposed hypothesis is confirmed, the project will significantly impact the country with the know-how generated.
Genetiği değiştirilmiş mikroorganizma kullanarak plastik degradasyonu
The usage of PET plastics in daily life have excessively increased in the last decade. The increased usage of PET is accompanied with the massive amount of PET waste accumulating rapidly. Environmental pollution caused by this waste has reached a critical point with pollutants being found even in the most remote parts of the world. Causing massive damage to ecosystems and even human health, PET plastic waste needs to be handled urgently. Although there are ongoing PET recycling and treatment efforts, the current methods in use are insufficient. The techniques currently used are either costly, leave a significant carbon footprint or are lacking in their ability to recycle microplastics. However, with the discovery of microorganisms which have the ability of degrading PET, biodegradation of PET products has emerged as a promising green alternative. In this thesis we designed bacterial tools to utilize the PET hydrolyzing enzyme, PETase. For this purpose, living bacterial platforms were engineered. The first system employed E. coli as the host to display PETase on the cellular surface. With PETase molecules anchored on its surface, aiding in the stability and the activity of the enzyme, the system will be a useful tool for PET degradation. For the surface display system, the Ag43 autotransporter protein is used. The system was cloned, and expression was analyzed using immunocytochemistry labeling. The activity of the system was analyzed with chromatography and mass spectrometry. The second system proposed uses E. coli once again as a workhorse for PETase secretion, creating a simple yet effective tool for the bioremediation of PET. For secretion of the enzyme, the disruption of Braun's lipoprotein to create a leaky outer membrane is exploited. The system was cloned, and the cloning was verified. Also, the activity of native PETase was analyzed with HPLC and mass spectrometry. With this analysis, the PET degrading activity of PETase was confirmed.
CRISPR tabanlı sentetik translasyonel regülasyonun konvansiyonel olmayan maya kullanılarak gerçekleştirilmesi
Efficient and programmable gene expression systems are essential for improving recombinant protein production in non-conventional yeast hosts such as Pichia pastoris. In this thesis, a synthetic gene regulation platform was established in P. pastoris by integrating rationally engineered GAP promoters with CRISPR/dCas9-based transcriptional activation modules. The aim was to convert the native constitutive GAP promoter into a tunable element capable of both activation and repression through gRNA-guided recruitment of effector domains, thus paving the way for orthogonal and context-specific control of gene expression. Two synthetic promoter variants (version 1 and version 2) were designed by introducing targeted mutations to create novel gRNA binding sites without disrupting core promoter function. These promoters were cloned upstream of an eGFP reporter and integrated into the genome of P. pastoris. Colony screening under various carbon sources (glucose, glycerol, ethanol, and methanol) revealed that most mutant promoters retained expression levels comparable to the wild-type PGAP, while certain clones displayed elevated eGFP production due to multiple gene integrations. Quantitative PCR analysis was employed to identify single-copy integrants for further use. Subsequently, a CRISPRa system comprising dCas9, MS2-binding scaffold RNAs, and the VP64 activation domain was introduced into selected single-copy clones. Ten custom-designed gRNAs (five for each promoter version) were tested under four carbon conditions to assess their activation potential. Notably, version 1 demonstrated robust transcriptional activation with specific gRNAs, especially v1-g2-c1, which significantly enhanced eGFP expression across all tested conditions. In contrast, version 2 failed to elicit notable activation, possibly due to unfavorable gRNA positioning or inhibitory mutations within the promoter sequence. This work introduces a modular and orthogonal transcriptional regulation system in P. pastoris, offering dynamic control over synthetic promoters using CRISPRa components. The approach establishes a foundation for future metabolic engineering strategies and recombinant protein expression systems that are independent of traditional inducible promoters and adaptable to various industrial contexts.
Sentetik biyoloji yaklaşımı ile anti-diyabetik yaşayan ilaç geliştirimi
Type 2 Diabetes Mellitus (T2DM) is a prevalent metabolic disorder characterized by insulin resistance and impaired glucose regulation. Peptide-based drugs such as GLP-1 and its analog Exendin-4 are widely used in clinical treatment due to their ability to enhance insulin secretion and improve glycemic control. However, frequent injections, enzymatic degradation in the gastrointestinal tract, and short half-life limit their therapeutic efficiency and patient compliance. To address these challenges, this study aims to develop a living therapeutic system that enables the dynamic, gut-responsive production of anti-diabetic peptides using engineered Escherichia coli Nissle 1917. In the first part of the study, whole-cell biosensors responsive to physiologically relevant stimuli such as fatty acids, bile salts, and aspirin were constructed using synthetic regulatory elements. These biosensors were initially characterized through the expression of a fluorescent reporter gene (sfGFP) to determine their dose-response behavior and functionality. Following successful characterization, the reporter gene was replaced with either GLP-1 or Exendin-4 coding sequences, fused to various signal peptides (PhoA, MalE, TorA, DsbA, PelB) to promote extracellular secretion. Gibson Assembly and classical cloning techniques were used throughout the construct designs. The functional activity of secreted peptides was evaluated through ELISA-based quantification and in vitro bioassays using MIN6 insulin-secreting cells. MTT assays were performed to assess cell viability, and glucose-stimulated insulin secretion assays were conducted to determine the biological activity of the secreted peptides. Among the tested constructs, signal peptide–fused versions of GLP-1 and Exendin-4 showed significant effects on cell viability and insulin secretion, indicating successful expression and functionality of the therapeutic peptides. This study demonstrates the feasibility of combining probiotic bacteria with metabolite-responsive gene circuits for targeted peptide delivery. The developed platform presents a promising strategy for designing next-generation living drugs capable of responding to the host environment and offering a self-regulated treatment for metabolic diseases like T2DM.
İnfluenza virüslerine yönelik hücresiz sentetik biyoloji destekli RHA toehold switch tanı sistemi
Influenza A H1N1 continues to pose a major public health threat due to its rapid transmission and capacity to cause seasonal epidemics and pandemic outbreaks. Rapid, accurate, and cost-effective diagnostic approaches are essential for timely intervention and control of viral spread. In this study, we present a synthetic biology-based diagnostic strategy that employs programmable RNA-based regulatory elements, known as toehold switches, to selectively detect Influenza A H1N1 viral RNA sequences. These switches were designed to remain translationally inactive in the absence of the viral RNA and to activate protein expression upon specific sequence recognition. The initial switch designs were computationally generated and analyzed using thermodynamic modeling tools such as NUPACK and RNAfold, allowing for assessment of structural stability and identification of high-entropy regions that could affect translation. Particular attention was given to the accessibility of the ribosome binding site and start codon regions, as local structural hindrances in these areas were found to correlate with poor performance. Based on the entropy profiles and free energy distributions, selected constructs were subjected to rational sequence redesign to enhance conformational accessibility and minimize undesired leakiness. These optimized switches were then cloned into T7 promoter-driven plasmids and tested through in vitro transcription–translation reactions. The resulting GFP-based fluorescence measurements allowed us to quantitatively compare expression levels in the presence and absence of the target RNA. Experimental data showed that optimized switch designs provided significantly higher signal-to-noise ratios, reduced background expression, and more consistent fold-change values across replicates compared to their unoptimized counterparts. Overall, this study demonstrates the potential of rationally engineered RNA-based switches as a modular, programmable, and low-cost diagnostic platform for Influenza A H1N1. Moreover, the design framework established here can be generalized to support the development of similar RNA-sensing tools for other viral pathogens.
Rekombinant konak platformu olarak lactobacillus plantarum'un mühendisliği
Lactobacillus plantarum is a versatile lactic acid bacterium recognized for its probiotic properties and long-standing use in fermented foods. While traditional microbial chassis such as E. coli or S. cerevisiae have dominated recombinant bioproduction platforms, their limitations in food-grade and probiotic applications necessitate the development of alternative hosts. L. plantarum, with its GRAS status, genetic malleability, and resilience under gastrointestinal conditions, has emerged as a promising next-generation chassis, especially for the biosynthesis of health-related metabolites. The aim of this study is to engineer L. plantarum WCFS1 as a recombinant host platform for the production of functional molecules, with an emphasis on 2′-fucosyllactose (2′-FL), a key human milk oligosaccharide known for its prebiotic and immunomodulatory benefits. To achieve this, advanced synthetic biology tools such as markerless CRISPR/Cas9 editing, auxotrophic selection strategies, and modular expression systems will be employed to introduce and regulate heterologous gene pathways responsible for 2′-FL biosynthesis. The project will also focus on overcoming strain-specific challenges such as transformation efficiency, plasmid stability, and metabolic balancing within the host. If successful, the engineered L. plantarum strain will serve as a live, food-compatible cell factory capable of producing 2′-FL either in fermenters or directly in situ as part of synbiotic formulations. The outcome of this work is expected to contribute to the growing field of probiotic metabolic engineering, offering a scalable, safe, and consumer-friendly platform for functional food innovation.
Biyomedikal uygulamalar için teşhis ve tedavi biyo-aracı
Biological systems are programmable by their nature. With using the abilities of these systems, scientists have designed, engineered and repurposed living machines for various tasks including biological sensing, recording of cellular events, drug production and disease treatment. Compared to the current methodology for these tasks, engineering biological systems provide a promising tool for the future of medicine, especially in the case of disease treatment. Type II Diabetes Mellitus (T2DM) is a medical condition which occurs by the deficiency of insulinotropic hormones inside the body and affects nearly half a billion people worldwide. Treatment strategies for this disease include monitoring patient for blood glucose levels, fine production of insulinotropic hormones and providing dose-controlled treatment for the patients. All these operations increase the cost of the treatment and cause a global problem for both medical professionals and patients. In this thesis, we propose novel systems for developing theranostic strategies for T2DM by using synthetic biology principles and genetically controlled sense-and-response cascades inside living cells. Proposed systems include a whole-cell glucose biosensor module, which can detect glucose concentrations by using internal glycolysis machinery of a probiotic Escherichia coli (E. coli) bacteria, and a release module, which can controllably secrete therapeutic molecules from the E. coli cell surface. To do that, we engineered an enzyme-based biosensor module which takes the pyruvate synthesized as a result of glycolysis and turns that molecule into hydrogen peroxide via SpxB pyruvate oxidase enzyme to later detect that signal with an optimized hydrogen peroxide biosensor. In order to later incorporate this biosensor with a release mechanism, we designed and engineered an Antigen-43 (Ag43) autotransporter based peptide release system. In that system, we used Ag43 autotransporter fused GLP-1 peptide, an insulinotropic hormone for the type II diabetes treatment that is controllably displayed on the cell surface. Another Ag43 fused protein, TEV protease, with a different control mechanism is also cooperated in the system to release GLP-1 from the surface by cutting the peptide from its recognition site. Taking the ability of glucose sensing and the successfully engineered release mechanisms, our proposed system has a huge potential to be used as an alternative system for treatment of the T2DM.
Genetiği değiştirilmiş hücreler ile biyomineralizasyonun sağlanması
Hydroxyapatite (HAP) is the final product of bone biomineralization process and HAP formation is controlled by proteins, enzymes and small molecules secreted to extracellular matrix (ECM). Among these molecules, alkaline phosphatase (ALP) leads formation of HAP crystals and noncollagenous proteins control crystal nucleation and growth, and inhibit crystal formation. Osteocalcin (OCN) and osteopontin (OPN), are the most abundant noncollagenous proteins in ECM, which controls mineralization events. In this study, effect of OCN and OPN on HAP crystal formation was studied in order to achieve controlled crystal growth. In vitro biomineralization assays were conducted to understand the effect of OCN and OPN on the crystal structure of as formed minerals. While OCN decreases crystal growth rate and inhibit mineralization, which leads to more uniform crystal formation, OPN provides faster mineral formation with reduced Ca/P ratio. Moreover, a mammalian engineered cell line was constructed to achieve expression of bone extracellular matrix (ECM) proteins. For this purpose, genetic cassettes were produced to express OCN and OPN proteins, which are the most common non-collagen proteins that control bone mineral formation. By this way, production of bone type minerals with controlled size, shape and Ca/P ratio can be possible. Our system provides a truly biomimetic approach to HAP formation compared to chemical synthesis methods in literature. We believe our current findings will lead to innovative approaches for bone biomineralization in regenerative medicine and bone tissue engineering.
Glikozilasyon: Protein tabanlı malzeme geliştirmede yeni bir araç
Biofilms are bacterial communities formed by adhesion of cells to each other via extracellular matrix. Proteins, one of the components of biofilm matrix, can form fibers and have properties enabling their use as materials. Bacillus subtilis biofilm major protein TasA is a fiber forming non-amyloidogenic protein providing biofilm rigidity. Glycosylation is a post-translational modification observed in all domains of life where sugar groups are added on proteins covalently. Campylobacter jejuni glycosylation is the first bacterial N-linked glycosylation discovered and the most studied system where a heptasaccharide is attached to protein. C. jejuni glycosylation has variety of functions among adhesion, protease resistance and thermal stability. Glycosylation may become an additional method for engineering biofilm proteins as materials with unique properties. For this purpose, we examined effect of glycosylation on structure of natively non-glycosylated proteins by glycosylating alkaline phosphatase enzyme (ALP) at different locations on the protein. Interestingly, phosphatase activity assay showed slight increase in ALP activity on pure proteins glycosylated at the C-terminus. Next, TasA protein was glycosylated at the C-terminus. Glycosylation had no significant effect on fibrillation of TasA in vitro. Secondary structure analysis using circular dichroism data revealed shift from antiparallel to helix structure with glycosylation. Quartz crystal microbalance experiments indicated increased adhesive properties on glycosylated TasA protein on gold. Its application possibility as cell adhesive was assessed by visualizing surface coverage of polystyrene cell culture plate under scanning electron microscope. As a consequence, glycosylation was used as an engineering method for protein-based material development for the first time.
Nanomalzeme kaynaklı toksisitenin gözlemlenmesi için tasarlanan sentetik gen devreleri
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.
Mikrobiyal amiloitlerin fonksiyonel biyomalzeme olarak kullanılması
Amyloids are fibrillar aggregations of proteins, dominated by β-sheets in the structure. They have high mechanical strength and rigidity that provides resistance to physical and chemical stress. In addition, amyloids can be easily functionalized with peptide groups for desired applications. These properties make them suitable candidates for biomaterial applications. Binding behavior of curli based amyloids on medically relevant surfaces are critical for their utilization controllable biomaterials. Furthermore, functionalization tor curli fibers too enhance their binding kinetics to surfaces and to organisms may hold great potentials for their desired applications as biomaterials. In this thesis, we characterized the binding kinetics of CsgA and CsgB curli proteins on silica, gold and hydroxyapatite surfaces to precisely control their surface adhesion. We showed that according to the physicochemical properties of surfaces, CsgA and CsgB displayed different binding behavior. Second, we hypothesized that glycosylation could enhance surface adhesiveness of curli fibers. For this purpose, TasA protein is engineered to obtain a glycosylation site and depicted that glycosylation increased adhesiveness of TasA fibers to gold surfaces. Finally, we functionalized CsgA curli fibers with RGD peptide to increase adhesiveness to living cells. Our results demonstrated that RGD peptide addition cause a significant increase in the number of cells adhered onto coated surfaces. In conclusion, amyloid proteins can serve as superior biomaterials with desired functionalities. Physicochemical properties of surfaces and proteins can have essential impacts on their interaction. In order to diversify those properties, amyloid fibers can be easily functionalized for specific purposes such as improved surface and cell adhesion. Functionalization with different peptide groups can extend their application capacity as superior biomaterials.
Escherıchıa colı bakterisinde protein glikolizlenmesi için devre tasarımı
Protein glycosylation is one of the most crucial and common post-translational modifications. Glycosylation provides certain advantages to host organisms and extend the proteome beyond genetic material. After the discovery of bacterial glycosylation mechanisms and especially after its transfer into laboratory work-horse E. coli, studies utilizing this mechanism increased exponentially. It has been previously showed that utilizing N-Linked Glycosylation, certain recombinant proteins have been furnished with improved features, such as stability and solubility. In this study, we utilized N-linked Glycosylation to glycosylate alkaline phosphatase (ALP) enzyme in E. coli and investigate the effects of glycosylation on an enzyme. Considering the glycosylation mechanism is highly dependent on the acceptor protein, ALP constructs carrying glycosylation tag at different locations of the gene has been created and glycosylation rates have been calculated. The most glycosylated construct has been selected for comparison with the native enzyme. Studies showed that glycosylated ALP performed better at optimal conditions. In order to extend the knowledge on the differences due to glycosylation, several conditions were applied. ii Both enzymes were tested at elevated temperatures for different incubation times, different pH conditions, protease treatment and under denaturing conditions. Also, secondary structure analysis was performed for each condition to elaborate on these differences. Experiments showed that glycosylated ALP performs remarkably better at all conditions tested. Therefore, N-linked Glycosylation mechanism can be employed for enzyme engineering purposes and is a useful tool for industrial applications that require enzymatic activity.
Fonksiyonel biyofilm proteinlerinin antibiyotik giderimi ve SARS-CoV-2 yakalanması için kullanımı
Among biomaterials, biofilm proteins occupy a great portion. They have many prominent properties such as mechanical strength, ability to be modified genetically and their stability against harsh physical and chemical conditions from their environment. Their high tendency to genetic modifications provides them wide application areas ranging from environmental pollution prevention to medical usage areas. Therefore, it is of crucial importance that biofilm proteins can handle different genetic modifications for a desired purpose of use. In this thesis, we aimed to form complex structures of CsgA biofilm protein with three different functional groups. The functional groups we used are laccase type enzymes, i.e. CotA and YlmD, and a lectin, Griffithsin (GRFT). The complex formation between CsgA biofilm protein and the aforementioned functional groups is achieved with the help of an irreversible bond formed when SpyTag-SpyCatcher protein domains interact with each other. With the complexes we obtained from CsgA and CotA, YlmD enzymes we performed degradation of a fluoroquinolone type antibiotic, which is abundantly found in the natural water bodies causing antibiotic resistance. The degradation products of the antibiotic were assessed via LCMS-QTOF. We have also formed a complex from CsgA and GRFT in which we aimed to capture SARS-CoV-2 virus particles from aqueous media. We have checked the infectivity of SARS-CoV-2 virus after incubation with the complex we created. In conclusion, CsgA biofilm protein can effectively be modified with various functional groups by making use of an irreversible chemical bond formed when a set of other proteins interact. The complex formed at the end can be used for different purposes such as pollutant degradation and virus capture. The complex system is prone to modifications with other functional groups for desired application areas.
Tümörleri hedeflemek için hücresel bir cihaz
Cancer is the second leading cause of death globally, affecting one out of three people during their lifetime. Due to its severity and high incidence, numerous treatment methods have been implemented, with the Chimeric Antigen Receptor T-Cell (CAR-T) therapy remaining the most promising one. Other therapy options such as surgery, chemotherapy and radiation therapy remain the backbone of cancer treatment, however these therapies are not effective enough as they do not discriminate among the healthy and cancerous tissues. Therefore, there is an imperative need in developing novel cancer treatment therapies that offer precise localization and on target therapeutics release. In this study, we aim to develop an engineered bacterial device, that can sense Jimt1 breast cancer cells, which are characterized by overexpression of human epidermal growth factor receptor 2 (HER2). 2Rs15d, a nanobody that binds to HER2 receptor, is expressed on the surface of Escherichia coli BL21 (DE3) via Ag43 autotransporter protein. Upon localization in the tumor site, a therapeutic agent will be released on the outer surface. By creating this platform, we aim to target the main problems of the existing cancer therapies.