Browsing by Subject "Synthetic biology"

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Open Access
A bacterial living therapeutics with engineered protein secretion circuits to eliminate breast cancer cells
(2024-01) Binte Shahid, Gozeel
Cancer therapy often faces limitations due to potential side effects, prompting scientific interest in bacteria-based living cancer treatments. Yet, the complete utilization of bacteria in therapeutic applications confronts engineering hurdles. This thesis focuses on introducing a novel bacterial mechanism specifically intended to target and eliminate breast cancer cells. Our innovative approach involves modifying Escherichia coli (E. coli) to secrete a Shiga toxin called HlyE, a pore-forming protein that binds to HER2 receptors found on breast cancer cells. This binding process is facilitated by a nanobody expressed on the bacterial surface through the Ag43 autotransporter protein system. Our research demonstrates the effective binding of the nanobody to HER2+ cells in laboratory conditions (in vitro). Utilizing the YebF secretion system, we successfully leverage the secretion of HlyE, leading to the eradication of the targeted cancer cells. These outcomes emphasize the significant potential of our engineered bacteria as an innovative and promising strategy for breast cancer treatment. This pioneering approach represents a groundbreaking development in the field of cancer therapeutics. By harnessing the unique properties of bacteria and utilizing advanced engineering techniques, we've succeeded in creating a targeted and potent system capable of attacking breast cancer cells specifically marked by the HER2 receptor. Our study lays a robust foundation for future exploration and development in the realm of bacterial-based cancer therapies, offering potential solutions to the challenges encountered in traditional cancer treatment methods.
Unknown
Biosystems engineering of prokaryotes with tumor-killing capacities
(Bentham Science Publishers Ltd., 2016) Kalyoncu, E.; Olmez, T. T.; Ozkan, A. D.; Sarioglu, O. F.
Certain bacteria selectively attack tumor tissues and trigger tumor shrinkage by producing toxins and modulating the local immune system, but their clinical utility is limited because of the dangers posed by systemic infection. Genetic engineering can be used to minimize the risks associated with tumor-targeting pathogens, as well as to increase their efficiency in killing tumor cells. Advances in genetic circuit design have led to the development of bacterial strains with enhanced tumor-targeting capacities and the ability to secrete therapeutics, cytotoxic proteins and prodrug-cleaving enzymes, which allows their safe and effective use for cancer treatment. The present review details the recent advances in the design and application of these modified bacterial strains.
Unknown
Biotechnological drug platforms
(2023-11) Ahan, Recep Erdem
Biopharmaceuticals, also known as biotechnological drugs, have revolutionized the treatment of many diseases by providing access to new mechanisms of action that can target the underlying biological processes behind the diseases. Technological advancement in biological sciences opens new paths to uncover new biopharmaceutical modalities in nature as well as to augment the existing modalities new functions. The implementation of engineering principles i.e., synthetic biology approaches have been transforming biopharmaceutical research wherein “smart” therapeutics are developed and deployed for treatment of previously intractable diseases. However, there are still unmet clinical needs that require novel and advanced biopharmaceuticals. In this thesis, I explored different biopharmaceuticals to characterize and/or advance their capabilities for diverse indications. Firstly, we have developed a prophylactic agent from the lectin protein, griffithsin, as for ancestral and the emerged strains of SARS-CoV-2. Secondly, we have advanced genetic technologies to engineer the probiotic Escherichia coli (E. coli) strain, Nissle 1917 (EcN), for therapeutical purposes. We developed a stable recombinant DNA transfer system based on cryptic plasmids of EcN. Furthermore, a synthetic protein secretion system was envisioned and functionally validated in EcN to shuttle therapeutical proteins to diseases site. Finally, peptide tags for extracellular protein secretion as well as a cell surface protein display system were developed for Lachnospiraceae species which are parts of the healthy human gut microbiome. The technologies and methodologies described herein will pay the way for inventing and/or discovering novel biopharmaceuticals to treat current and future diseases.
Open Access
Cellular biocatalysts using synthetic genetic circuits for prolonged and durable enzymatic activity
(Wiley‐VCH Verlag GmbH & Co. KGaA, Weinheim, 2019) Ahan, Recep Erdem; Şaltepe, Behide; Apaydın, Onur; Şeker, Urartu Özgür Şafak
Cellular biocatalysts hold great promise for the synthesis of difficult to achieve compounds, such as complex active molecules. Whole‐cell biocatalysts can be programmed through genetic circuits to be more efficient, but they suffer from low stability. The catalytic activity of whole cells decays under stressful conditions, such as prolonged incubation times or high temperatures. In nature, microbial communities cope with these conditions by forming biofilm structures. In this study, it is shown that the use of biofilm structures can enhance the stability of whole‐cell biocatalysts. We employed two different strategies to increase the stability of whole‐cell catalysts and decrease their susceptibility to high temperature. In the first approach, the formation of a biofilm structure is induced by controlling the expression of one of the curli component, CsgA. The alkaline phosphatase (ALP) enzyme was used to monitor the catalytic activity of cells in the biofilm structure. In the second approach, the ALP enzyme was fused to the CsgA curli fiber subunit to utilize the protective properties of the biofilm on enzyme biofilms. Furthermore, an AND logic gate is introduced between the expression of CsgA and ALP by toehold RNA switches and recombinases to enable logical programming of the whole‐cell catalyst for biofilm formation and catalytic action with different tools. The study presents viable approaches to engineer a platform for biocatalysis processes.
Open Access
Cellular biosensors with engineered genetic circuits
(American Chemical Society, 2018) Saltepe, Behide; Kehribar, Ebru Şahin; Yirmibeşoǧlu, Side Selin Su; Şeker, Urartu Özgür Şafak
An increasing interest in building novel biological devices with designed cellular functionalities has triggered the search of innovative tools for biocomputation. Utilizing the tools of synthetic biology, numerous genetic circuits have been implemented such as engineered logic operation in analog and digital circuits. Whole cell biosensors are widely used biological devices that employ several biocomputation tools to program cells for desired functions. Up to the present date, a wide range of whole-cell biosensors have been designed and implemented for disease theranostics, biomedical applications, and environmental monitoring. In this review, we investigated the recent developments in biocomputation tools such as analog, digital, and mix circuits, logic gates, switches, and state machines. Additionally, we stated the novel applications of biological devices with computing functionalities for diagnosis and therapy of various diseases such as infections, cancer, or metabolic diseases, as well as the detection of environmental pollutants such as heavy metals or organic toxic compounds. Current whole-cell biosensors are innovative alternatives to classical biosensors; however, there is still a need to advance decision making capabilities by developing novel biocomputing devices.
Open Access
Combating infectious diseases with synthetic biology
(American Chemical Society, 2022-01-25) Khan, Anooshay; Ostaku, Julian; Aras, Ebru; Safak Seker, Urartu Ozgur
Over the past decades, there have been numerous outbreaks, including parasitic, fungal, bacterial, and viral infections, worldwide. The rate at which infectious diseases are emerging is disproportionate to the rate of development for new strategies that could combat them. Therefore, there is an increasing demand to develop novel, specific, sensitive, and effective methods for infectious disease diagnosis and treatment. Designed synthetic systems and devices are becoming powerful tools to treat human diseases. The advancement in synthetic biology offers efficient, accurate, and cost-effective platforms for detecting and preventing infectious diseases. Herein we focus on the latest state of living theranostics and its implications.
Open Access
Design and applications of self-assembled soft living materials using synthetic biology
(Elsevier, 2022-01-01) Özkul, Gökçe; Yavuz, Merve; Hacıosmanoğlu, Nedim; Kırpat, Büşra Merve; Şeker, Urartu Özgür Şafak
In nature, the cells are unique biofactories of various kinds of macroscale structures. These biofactories are as old as the earth. However, as technology developed and new areas of research fields developed these cellular biofactories became the center of attention. The motive was the question if we can engineer them according to the world’s needs. At that point, approaches and tools of synthetic biology came into the picture. After its development, people started to engineer biofactories and produce materials with new properties. One of those materials is classified as self-assembled soft living materials with their specific features and usage areas. To be more specific, biofilms are examples of self-assembled soft living materials due to their self-sustaining and self-assembling properties. They can be engineered starting from genetic circuits leading to creation of their building blocks and finally formation of complex biofilm systems. With the diversity in their engineering aspects, their application areas also vary. In this chapter, the design of biofilm structures from genetic circuits until the formation of complex biofilm structures and their various applications will be investigated.
Open Access
Design of synthetic biological devices for detection and targeting human diseases
(Elsevier, 2022-01-01) Hacıosmanoğlu, Nedim; Köse, Sıla; Ostaku, Julian; Köksaldi, İlkay Çisil; Saltepe, Behide; Şeker, Urartu Özgür Şafak; Singh, V.
Interpreting signals coming from the surrounding environment and responding to these stimuli by adjusting physical or metabolic state is the most fundamental ability of living organisms. Repurposing these natural abilities for the detection and responding to different molecules is one of the key focuses of synthetic biology because the overall strategy could provide advanced solutions for different diseases. Also by being naturally suitable to the design-build-test-learn manner of synthetic biology, biosensors are great examples of what engineering and biology could achieve when they come together. Literature has many examples of intellectually designed biosensor systems, which may overachieve and outperform existing technologies with a completely biocompatible structure. In this chapter, design and application of these biosensor systems will be investigated.
Open Access
Engineered bacteria with genetic circuits accumulating nanomagnets as MRI contrast agents
(Wiley, 2022-01-25) Yavuz, Merve; Ütkür, Mustafa; Kehribar, Ebru Şahin; Yağız, Ecrin; Sarıtaş, Emine Ülkü; Şeker, Urartu Özgür Şafak
The demand for highly efficient cancer diagnostic tools increases alongside the high cancer incidence nowadays. Moreover, there is an imperative need for novel cancer treatment therapies that lack the side effects of conventional treatment options. Developments in this aspect employ magnetic nanoparticles (MNPs) for biomedical applications due to their stability, biocompatibility, and magnetic properties. Certain organisms, including many bacteria, can synthesize magnetic nanocrystals, which help their spatial orientation and survival by sensing the earth's geomagnetic field. This work aims to convert Escherichia coli to accumulate magnetite, which can further be coupled with drug delivery modules. The authors design magnetite accumulating bacterial machines using genetic circuitries hiring Mms6 with iron-binding activity and essential in magnetite crystal formation. The work demonstrates that the combinatorial effect of Mms6 with ferroxidase, iron transporter protein, and material binding peptide enhances the paramagnetic behavior of the cells in magnetic resonance imaging (MRI) measurements. Cellular machines are also engineered to display Mms6 peptide on the cell surface via an autotransporter protein that shows augmented MRI performance. The findings are promising for endowing a probiotic bacterium, able to accumulate magnetite intracellularly or extracellularly, serving as a theranostics agent for cancer diagnostics via MRI scanning and hyperthermia treatment. © 2022 Wiley-VCH GmbH.
Open Access
Engineering of probiotic Saccharomyces boulardii as a host for living therapeutic applications
(2022-09) Kurt, Orhan Nedim
Therapeutic molecules or biologically active agents used in the treatments of disorders have been manufactured employing chemical synthesis methods with high costs and limited accessibility. Even though this approach to producing therapeutic molecules has enabled life-changing substances to be produced, there are still countless diseases waiting for a cure. Therefore, it would not be possible to meet the treatment needs via just conventional manufacturing approaches. Living therapeutics are programmed cells for the production and direct delivery of therapeutic molecules to the human body. Indeed, biologically engineered cells have been used for a long time to manufacture biomolecules in industrial settings. Also, there have been many studies that designed and programmed living organisms to cure diseases such as cancer, diabetes, inflammatory bowel syndrome, ulcer, and colitis. accharomyces boulardii CNCM I-745 is a yeast strain used as a probiotic for a very long time in humans. S. boulardii is the first and the only yeast strain approved to be used in human medicine so far. It has been shown that S.boulardii has many benefits to its host, including restoring the microbiome and competing with pathogens. Also, S.boulardii is a promising organism to be engineered and programmed as a living therapeutic factory due to its great compatibility with the human body and its metabolic features enabling us to manufacture complex molecules. Aiming for this, we created a TRP1 auxotroph S.boulardii strain using synthetic biology tools to eliminate the use of antibiotic resistance genes in the further steps since it would be problematic for the spread of antibiotic resistance. To do that, we employed the CRISPR CAS-9 system and a donor DNA harboring a stop codon to be inserted into the target gene (TRP1). After verifying the construction of the TRP1 auxotroph strain, we designed an episomal expression vector comprising a strong, constitutive promoter, extracellular signal sequence (Alpha sequence), and a terminator (CYC1). Next, we inserted the Intrinsic Factor protein (the key protein responsible for vitamin B12 absorption) sequence into the vector to recombinantly produce it and secrete it into the gut. However, according to our preliminary data, we could not observe an expression of the protein of interest, suggesting that the system needs further optimization and investigation to work properly. Considering the lack of auxotrophic yeast strains available to be engineered for therapeutic purposes, our engineered S.boulardii strain can be employed in yeast-based living therapeutics applications against different disorders waiting for their treatment.
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
Genetically designed microbes for bioimaging and biosensing
(2024-09) Yavuz, Merve
The advantageous approach to the utilization of the microbes for bioimaging and biosensing underlies under their active motility and self-propulsion characteristics besides their easy bioengineering feature to gain multi-functional activities. The emerging developments make use of microorganisms as therapeutic agents in disease diagnosis and treatment. The dynamic nature of the habitat forces the microorganisms to acclimate themselves to changing living conditions via evolving exclusive bio-functionalities for their survival. Therefore, the living microorganisms producing functional materials serve as a biohybrid system with unprecedented potential for enhancing the detection of a disease biomarker molecule or meeting the great need in cancer diagnosis. The synthetic biology approach, a multidisciplinary field of science, gives the ability to engineer and modulate the microorganisms to redesign existing natural pathways, resulting in the gain of the desired function. Inspiring form nature, the biomineralization of iron-oxide materials is demanding for their potential usage in antitumor effect due to their easy modulation, stability, and magnetic properties. Furthermore, the certain respiratory capacities of electrochemically active microbes enable the respiration of diverse inorganic and organic molecules for their survival in redox-stratified environments. The ability of exchanging electrons with electrodes possesses several diverse biotechnological applications like the construction of microbial fuel cells, electro-fermentation, and electro-genetics. In this thesis, the microbes were engineered for their utilization in bioimaging and biosensing applications. Firstly, intracellular and extracellular magnetite accumulating Escherichia coli bacterial cell machineries were constructed as contrast agents for the MRI scanning, promising for a cancer diagnostic. Secondly, the intracellular magnetite accumulating bacterial cells, possessing all the redox reactions that readily take place in their cytoplasm via synthetically produced proteins, were further engineered to improve their targeting capability for breast cancer tumor cells by displaying a certain nanobody on the cell surface. Thirdly, electronic sentinel bacterial cells were designed utilizing the electron transfer modules for extracellular electron consumption by targeted acceptors for their wireless biomonitoring applications upon detecting a disease molecule. The methodologies described in this thesis are envisioned as promising tools for diagnostic applications.
Open Access
A living material platform for the biomineralization of biosilica
(Elsevier B.V., 2022-12-15) Kırpat Konak, Büşra Merve; Bakar, Mehmet Emin; Ahan, Recep Erdem; Özyürek, Emel Uzunoğlu; Dökmeci, Serap; Şafak Şeker, Urartu Özgür
Nature has a vast array of biomineralization mechanisms. The commonly shared mechanism by many living organisms to form hardened tissues is the nucleation of mineral structures via proteins. Living materials, thanks to synthetic biology, are providing many opportunities to program cells for many functionalities. Here we have demonstrated a living material system for biosilicification. Silaffins are utilized to synthesize silicified cell walls by one of the most abundant organism groups called diatoms. The R5 peptide motif of the silaffins is known for its ability to precipitate silica in ambient conditions. Therefore, various studies have been conducted to implement the silicification activity of R5 in different application areas, such as regenerative medicine and tissue engineering. However, laborious protein purification steps are required prior to silica nanoparticle production in recombinant approaches. In this study, we aimed to engineer an alternative bacterial platform to achieve silicification using released and bacteria-intact forms of R5-attached fluorescent proteins (FP). Hence, we displayed R5-FP hybrids on the cell surface of E. coli via antigen 43 (Ag43) autotransporter system and managed to demonstrate heat-controllable release from the surface. We also showed that the bacteria cells displaying R5-FP can be used in silicification reactions. Lastly, considering the stimulating effect of silica on osteogenic differentiation, we treated human dental pulp stem cells (hDPSCs) with the silica aggregates formed via R5-FP hybrids. Earlier calcium crystal deposition around the hDPSCs was observed. We envision that our platform can serve as a faster and more economical alternative for biosilicification applications, including endodontics. © 2022
Open Access
Making the next generation of therapeutics: mRNA meets synthetic biology
(American Chemical Society, 2023-09-15) Hınçer, Ahmet; Ahan, Recep Erdem; Aras, Ebru; Şeker, Urartu Özgür Şafak
The development of mRNA-based therapeutics centers around the natural functioning of mRNA molecules to provide the genetic information required for protein translation. To improve the efficacy of these therapeutics and minimize side effects, researchers can focus on the features of mRNA itself or the properties of the delivery agent to achieve the desired response. The tools considered for mRNA manipulation can be improved in terms of targetability, tunability, and translatability to medicine. While ongoing studies are dedicated to improving conventional approaches, innovative approaches can also be considered to unleash the full potential of mRNA-based therapeutics. Here, we discuss the opportunities that emerged from introducing synthetic biology to mRNA therapeutics. It includes a discussion of modular self-assembled mRNA nanoparticles, logic gates on a single mRNA molecule, and other possibilities.
Embargo
Multiplexed cell-based diagnostic devices for detection of renal biomarkers
(Elsevier, 2022-12-24) Köse, Sıla; Ahan, Recep Erdem; Köksaldı, İlkay Çisil; Olgaç, A.; Kasapkara, Çiğdem S.; Şeker, Urartu Özgür Şafak
The number of synthetic biology-based solutions employed in the medical industry is growing every year. The whole cell biosensors being one of them, have been proven valuable tools for developing low-cost, portable, personalized medicine alternatives to conventional techniques. Based on this concept, we targeted one of the major health problems in the world, Chronic Kidney Disease (CKD). To do so, we developed two novel biosensors for the detection of two important renal biomarkers: urea and uric acid. Using advanced gene expression control strategies, we improved the operational range and the response profiles of each biosensor to meet clinical specifications. We further engineered these systems to enable multiplexed detection as well as an AND-logic gate operating system. Finally, we tested the applicability of these systems and optimized their working dynamics inside complex medium human blood serum. This study could help the efforts to transition from labor-intensive and expensive laboratory techniques to widely available, portable, low-cost diagnostic options.
Open Access
Peptide based ligand discovery to prevent protein aggregation in neurodegenerative disease conditions
(2019-09) Beğli, Özge
Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease are cognitively and physically debilitating and progressive diseases due to the gradual and irreversible loss of discrete neuronal populations in the brain. In addition to millions of people worldwide suffering from them, the prevalence of the neurodegenerative diseases dramatically increases with the increasing lifespan of the population. Most of the current therapeutic strategies either target toxic aggregates in neurons or support the healthy cells in diseased region. However, these interventions provide only symptomatic relief and deceleration of disease progression. Besides, aggregation involves a locking phase in which irreversible transition of soluble monomeric and oligomeric molecules into insoluble fibrous structures occurs. During aggregation, fragmentation of mature fibrils leads to the formation of new oligomeric structures possessing seeding activity. The seeds behaving as a nucleation unit trigger other structures to join the accumulated proteins. Synthetic biology is an emerging field that suggests therapeutic solutions for several diseases. Development of synthetic proteins such as artificial transcription factors and improved antibodies, artificial cell transplants with controlled secretion, designed inhibitory RNA molecules and antisense oligonucleotides, gene circuits and logic gates, synthetic viruses as an advanced delivery system and genome editing technologies using programmable nucleases are revolutionary approaches for the diagnosis and treatment of diseases. With the utilization of a variety of advanced tools, synthetic biology is extremely promising to treat neurodegenerative disorders too. In this study, biotechnological approaches and tools such as gene cloning, yeast surface display and phage display library have been used to target neurodegenerative proteins before aggregation takes place. Neurodegenerative proteins were cloned into a plasmid DNA within bacteria and displayed on the surface of Saccharomyces cerevisiae cells. A phage display library has been screened against those neurodegenerative proteins and binding peptides of these proteins have been selected following recursive rounds of binding and washing steps. Peptides that bind to neurodegenerative proteins with high affinity possess the potential to block them and prevent the initiation of aggregation. Beside to the promising results of neuroprotective and neurorestorative interventions, this strategy can provide prevention of aggregation which is the underlying cause of neurodegeneration.
Open Access
A recombinase-based genetic circuit for heavy metal monitoring
(MDPI, 2022-02-16) Akboğa, Doğuş; Saltepe, Behide
Rapid progress in the genetic circuit design enabled whole-cell biosensors (WCBs) to become prominent in detecting an extensive range of analytes with promise in many fields, from medical diagnostics to environmental toxicity assessment. However, several drawbacks, such as high background signal or low precision, limit WCBs to transfer from proof-of-concept studies to real-world applications, particularly for heavy metal toxicity monitoring. For an alternative WCB module design, we utilized Bxb1 recombinase that provides tight control as a switch to increase dose-response behavior concerning leakiness. The modularity of Bxb1 recombinase recognition elements allowed us to combine an engineered semi-specific heat shock response (HSR) promoter, sensitive to stress conditions including toxic ions such as cadmium, with cadmium resistance regulatory elements; a cadmium-responsive transcription factor and its cognitive promoter. We optimized the conditions for the recombinase-based cadmium biosensor to obtain increased fold change and shorter response time. This system can be expanded for various heavy metals to make an all-in-one type of WCB, even using semi-specific parts of a sensing system. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.
Open Access
A synthetic biology approach for nanomaterial design, synthesis and functionalization
(2017-11) Ölmez, Tolga Tarkan
Biological formation of inorganic material occurs in most organisms in nature. Various biomolecules such as polypeptides, lipids and metabolites are responsible for biomineralization in cells and tissues. Biological synthesis of biohybrid materials is a recently emerged discipline that uses these biomolecules in synthetic biological systems. Synthetic biology is one of most promising approaches for the development of biohybrid systems, and stands at the intersection of computer science, engineering and molecular genetics. Synthetic biology tools allow the design of programmable genetic toolkits that can compete with natural biosynthesis systems. The present thesis elaborates on the formation of well-controlled genetic systems that can synthesize and functionalize biological materials. Artificial peptides were fused to various genes through molecular genetics techniques, allowing the production of designer proteins. One aspect concerns the fusion of the 19 amino acid-long R5 motif of silaffin protein to three distinct fluorescent proteins. The R5 peptide motif can nucleate silica precursor ions to synthesize silica nanostructures. Therefore, fusion of fluorescent proteins with the R5 motif allows the synthesis and encapsulation of fluorescent silica nanoparticles. Due to its affinity to silica, R5 tag was also shown to be a candidate tag for silica resin-based affinity chromatography purification. Using synthetic biology tools, production of autonomously formed biotemplating platforms can be achieved. A bacterial functional amyloid fiber biosystem called curli can be utilized as a biotemplating platform for nanomaterials synthesis in this context. The major curli subunit CsgA was fused to artificial peptides that can nucleate and synthesize various nanomaterials. Inducible systems were also integrated into the genetic design system to confer temporal control over curli synthesis. These designs were improved through the incorporation of material-sensitive transcription factors and their cognate promoters for ions of cadmium, gold and iron. First, these material sensitive pairs were used in the development of microbial whole cell sensors that produce a fluorescence output upon induction by material precursor ions. Later, material-sensitive pairs were integrated into a modified curli nanofiber display biosystem to produce living autonomous whole cell nanomaterial synthesizers. These systems recognize precursor ions in the environment and synthesize modified curli nanofibers that can nuclate precursor ions to form functional nanomaterials.
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.