LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans
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ÖgeInvestigation of the effect of valproic acid, an hdac inhibitor, on the relationship between oxidative stress and autophagy in human eosinophil(Graduate School, 2022)Epigenetics bridges the gaps between phenotype and genotype. Any process that modulates the gene activity without causing any change in DNA sequence is called epigenetic modification. Epigenetic modifications are divided into three main groups: histone modifications, DNA methylation, and non-coding RNAs. These changes are reversible because they do not alter DNA sequence. However, dysregulation in modifications can lead to several diseases such as cancer, metabolic, and neurological disorders. Inhibitors that suppress modifications such as DNA methylation, histone deacetylase, histone acetyl transferase, and protein methyltransferase have been developed to reverse the epigenetic processes that drive the pathogenesis of diseases and are used as epigenetic drugs for treatments. One of the most widely used inhibitors in therapy is histone deacetylation inhibitors (HDACi). Valproic acid (VPA), a branched short-chain fatty acid obtained from valeric acid, is one of the drugs used as HDACi. VPA is generally used as an anticonvulsant drug for the effective treatment of different types of diseases such as epileptic seizures, migraine headaches, bipolar disorder. In addition, VPA has the promising potential to be effective in cancer therapy and controlling allergic responses because it can mediate the expression of important genes involved in anti-tumor immunity and activation of cells. Nevertheless, even when VPA is used therapeutically, studies have revealed that VPA induces oxidative stress, and can activates autophagy pathway, inflammasome response in different cells as a side effect. In this thesis, our aim is primarily to investigate the effects of valproic acid-induced stress on the activation of the antioxidant pathway, inflammasome complexes, and autophagy pathways that maintain cell balance in Eol-1 cells. Eol-1 human eosinophilic cells were used in this study since eosinophils have a key effector role in the response to allergic reactions. When the balance of reactive oxygen species (ROS) production in the cell is disrupted, oxidative stress arises in cells. ROS are highly reactive molecules and, elevated levels of ROS can damage cell compounds. Therefore, regulation of reactive oxygen species (ROS) generation is crucial for the proper functioning of cells. To maintain homeostasis, cells develop a series of antioxidant responses to reduce the toxicity caused by oxidative stress. In this case, the main regulator in the cell is the antioxidant transcription factor nuclear factor erythroid 2-associated factor 2 (Nrf2). Under normal conditions, Nrf2 interacts with Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm and leads to degradation of Nrf2 by the 26s proteasome, thus it is a negative regulator of Nrf2. Oxidative stress causes a conformational change in Keap1, leading to the dissociation of Nrf2 and Keap1. This enhances the acetylation and nuclear translocation of Nrf2. Proteins which transcribed by Nrf2 are involved in the activation of both anti-inflammatory and pro-inflammatory pathways in the cell to inhibit excessive immune responses caused by oxidative stress. Our results showed that VPA stimulation increased the cellular ROS formation in a dose-dependent manner at 24 h post-stimulation without any change in viability of Eol-1 cells. Moreover, the activation marker CD69 was upregulated via VPA treatment in dose-dependent manner. At lower concentrations, VPA augmented the protein level of Nrf2 and acetylated Nrf2, while attenuating the protein level of Keap1 in a dose-dependent manner. The immune system represents cells, tissues, organs, and components that come together to form a defense network to fight various pathogens or diseases by the excessive reaction and invasion of the microorganisms and to maintain the homeostasis of the host. The immune system consists of two arms: innate immunity and adaptive immunity. The first defense against pathogens is provided by innate immunity and white blood cells such as granulocytes (basophils, mast cells, eosinophils, neutrophils), monocytes, and Langerhans cells which are the main effector cells in innate immunity. The innate immune response is controlled by membrane/cytoplasmic receptors, inflammatory proteins, secreted cytokines, and chemokines. Pattern recognition receptors (PRRs) are mainly categorized as Toll-like receptors (TLR), NOD-like receptors (NLR), C-type Lectin (CLR) and RIG-I-like receptors (RLR). They can recognize different pathogen associated molecular patterns (PAMPs) and danger associated molecular patterns (DAMPs). Ligand-receptor interaction initiates inflammatory responses. The major canonical inflammatory responses are driven by NLRP3, NLRC4 inflammasome complexes against microbial structures and danger singals such as bacteria, mitochondrial ROS, and ATP. The NLRP3 and NLRC4 proteins combine with ASC and caspase 1 separately to form complexes. Activated caspase-1 cleaves pro forms of IL-1B, IL-18, and gastermin D (GSDMD) into mature forms. Firstly, GSDMD oligomerizes, then migrates to the membrane to form pores, so mature cytokines can be released from the pores. Cell membrane integrity is disrupted, and cells eventually die; this process is called pyroptosis. The release of cytokines and chemokines induces the migration and activation of many inflammatory cells, which is important for the proper immune response in microbial defense. According to our findings VPA treatment reduced the protein level of NLRC4 in a dose dependent fashion, but interestingly NLRP3 and caspase-1 cleavage, cleaved IL1β protein levels were elevated at lower doses in Eol-1 cells. IL-1B cleavage increased in parallel to the activity of caspase-1 and the inflammasome complex. Although cleaved IL1B was increased intracellularly, IL-1B and IL-10 secretion via VPA stimulation did not significantly change as compared to non- treated group. Autophagy is a mechanism responsible for maintaining homeostasis in the cell under various stress conditions. Cells remove damaged and unnecessary cell components through lysosomal degradation in response to cellular stresses such as nutrient deficiency or high levels of reactive oxygen species (ROS). As the most studied autophagy type, in macroautophagy, cytoplasmic components merge with the lysosome with the help of vesicles called autophagosome, forming autolysosome structures and the components inside the autophagosome are degraded. Various genes/proteins and pathways are involved in the maintenance of autophagy. The MAPK (mitogen-activated protein kinase), Akt (alpha serine/threonine-protein kinase) and mTOR (mammalian target of rapamycin) pathways are in the upstream of the autophagy pathway and are responsible for the regulation of cell growth, cellular metabolism, cell survival, and proliferation of cells. ERK1/2 is phosphorylated in the presence of autophagosome and thus it can be used as a positive marker in the autophagy pathway. Conversely, Akt and mTOR pathways negatively regulate the autophagy pathway. Phosphorylation of the Akt signaling pathway affects activation of the mTOR pathway as mTOR is downstream of Akt. In addition to these, microtubule‑associated protein light chain 3 (LC3) is the main marker in the autophagy flux. The cytosolic form LC3-I is converted to the LC3-II form through autophagy activation. LC3-II binds to the inner and outer membrane of the autophagosomes and preautophagosomal structure (PAS). Also, Beclin 1 is used as a marker for autophagy activation because it is crucial for the autophagosome structure and suppresses mTOR activity while inducing ERK activity. Our findings indicated that following VPA treatment, p44/42 MAPK (ERK1/2) protein increased at ascending doses, and phosphorylation of p44 MAPK was upregulated at lower doses. On the other hand, while the protein level of Akt didn't change after VPA, phosphorylation of Akt on both serine 473 and threonine 308 was downregulated via VPA stimulation. Moreover, the mTOR protein levels and the phosphorylation of mTOR decreased at high VPA doses, while protein levels of LC3B II was increased by VPA stimulation. However, increasing concentrations of VPA decreased the Beclin-1 protein level. Our results suggest that VPA stimulation induced the autophagy pathway independently of Beclin1in Eol-1 cells. Based on our current data, VPA led to oxidative stress by increasing ROS production levels and activated the antioxidant transcription factor NRF2 in Eol-1 cells. Also, VPA activated Eol-1 cells. VPA induced inflammasome complex formation without any change in the secretion of proinflammatory cytokines. Finally, VPA activated the autophagy pathway independently of Beclin1 in Eol-1 cells. Our results demonstrated the side effects that should be considered in the therapeutic use of VPA. In addition, the fact that VPA increases the activation of Eol-1 cells raises the question of whether VPA can not be used as a drug in allergic responses since it is not desired to increase the severity of the reaction in allergic responses.
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ÖgeRecombinant production and characterization of aquaporin protein isolated from geobacillus thermoleovorans ARTR1 and virgibacillus sp. agtr strains(Graduate School, 2022)Cells have a fluid mosaic model that provides the passage of organic and inorganic molecules, ions and water, which are survival for the cell, that form the structure of cell membranes and contain protein, carbohydrates and cholesterol. Proteins located in the membrane of the cell are called biological membrane proteins and they constitute approximately 25-30 % of all proteins. However, the partially hydrophobic surfaces of membrane proteins are still a largely unconquered area due to their lack of flexibility and stability. Biological membrane proteins have three subgroups depending on their location are analyzed in category. Integral membrane proteins, one of the biological membrane proteins which are permanently attached to the cell membrane. Detergents, non-polar solvents, or sometimes denaturing agents are used to separate this protein from the biological membrane. Integral membrane proteins are a permanent part of the membrane and can penetrate the membrane to form transmembrane proteins. Peripheral membrane proteins, another biological membrane protein, temporarily bind to the membrane or integral membrane proteins by hydrophobic, electrostatic, and other non-covalent interactions. These proteins can dissociate from the membrane after treatment with a solution containing a high pH or high salt concentration. Lipid-anchored proteins, on the other hand, are proteins on the surface of the cell membrane that covalently bind to lipids embedded in the cell membrane. Today, the structural and functional studies of the above-mentioned biological membrane proteins have been largely hampered by the difficulties in their production and overexpression, which are necessary for the structural studies of membrane proteins. Compared to other protein classes, the determination of the structure, expression, and purification of membrane proteins is greatly hampered by difficulties. Aquaporin, a biological membrane protein of great medical and industrial importance, are integral proteins that form pores in cell membranes and facilitate the transport of water between cells. This protein is the membrane protein that allows the passage of water molecules and rejects all other solutes. In the literature, there is a number of researches on the discovery of aquaporin from different cell membranes.While aquaporins, which are at the forefront of biomimetic membrane production in prokaryotic cells in the industrial area, are known for the passage of molecules that provide the vital conditions of the cell in plant cells and their necessity during some stress conditions (drought), in mammalians aquaporins are associated with many diseases, especially leukemia. The biggest difficulty encountered during the studies carried out in this direction is the obstacles in ensuring the overexpression of the integral biological membrane protein aquaporins and the optimization of their purification afterwards. Aquaporins have a similar basic structure: aquaporin monomers consist of six transmembrane helical segments and two short helical segments surrounding cytoplasmic and extracellular vestibules connected by narrow aqueous pore. Between the helices are five regions (A-E) with an asparagine-proline-alanine ("NPA motif") pattern, two of which are hydrophobic (B, E) that fold in or out of the cell membrane. Another part of the channel is the "ar/R selectivity filter", a set of amino acids that allows aquaporin to selectively allow or block the passage of different molecules. Aquaporin monomers can assemble as tetramers in membranes, with each monomer functioning independently. The primary function of most aquaporins is to transport water across cell membranes in response to osmotic gradients created by active solute transport. Aquaporins provide a 10-100-fold increase in water transfer across the cell membrane. Because of their unique properties, they need to be produced in large quantities for different purposes, including structure and activity analysis or new biomimicry materials. Different species of aquaporins may have different permeability and rejection properties for the same solute. While aquaporin may vary depending on solute concentrations, the selectivity of aquaporin also varies depending on load and size. It creates a positively charged energy barrier due to the amino acid Arginine in its structure. In this way, aquaporin prevents the passage of loaded or neutral solvents. Over the past 5 years, there has been great interest in the biology of aquaporins (AQPs) in over 500 studies published on aquaporin cloning, genetics, tissue localization, developmental and regulated expression, transgenic mouse models, and structure/function analyzes. When aquaporins, which are found in all cells from bacteria to mammals, are investigated in the literature, it is seen that mostly AQP0, AQP1, AQP2, AQP4, AQP5, AQP6 and AQP8 species are water specific. AQP3, AQP7 and AQP9 are called aquaglyceroporins and also carry glycerol and/or other small uncharged molecules. Most studies on recombinant aquaporins have so far been functional, regulatory or structural studies of aquaporins. In addition to these studies, the application of aquaporins with high water permeability and high solute rejection with biomimetic membranes for water desalination and reuse has attracted great interest in recent years. Prokaryotic aquaporins were first identified in Escherichia coli, and microorganisms such as Rhodobacter sphaeroides, Lactococcus lactis, Saccharomyces cerevisiae, Mathanothermobacter marburgensis, Photobactetrium profundum SS9 and Halomonas elongate have been used in studies to purify aquaporin protein. Also the recombinant production of aquaporin is being studied by using cell-free expression systems. Studies with prokaryotic AQPs show that bacteria help cope with osmotic, oxidative stress and nutritional fluctuations. However, there are limited studies in the literature for aquaporins produced from prokaryotic organisms and especially prokaryotes under extreme conditions. Virgibacillus sp. AGTR strain isolated from Acıgöl (Burdur, Turkey), which is a lake with high salinity has taken its place in the literature as a new halophilic bacterium strain. The genome information of this organism was obtained, and it was determined that it contains two aquaporin-encoding genes, one of which is 831 bp and the other 795 bp. Also, a new thermophilic bacterium Geobacillus thermoleovorans ARTR1 was isolated from another extreme environment, Armutlu (Yalova). The gene sequence encoding aquaporin with a length of 819 bp was determined in this organism. To analyze the stability of extremophilic aquaporins in industrial processes requiring harsh conditions (high temperature, high pH), in total, three extremophilic aquaporin genes were isolated from both halophilic Virgibacillus sp. AGTR and thermophilic Geobacillus thermoleovorans ARTR1 strains. These isolated genes were transferred to the pET28a (+) vector and produced recombinantly in the Escherichia coli C43 host cell. In this process, genes related to the PCR method with appropriate primers were amplified and ligated with a pET28a (+) vector. IPTG induction was subjected to time, temperature, and molarity trials, respectively. Then the optimal condition of 1 mM IPTG, 37 °C and 4 hours induction was selected. Under this optimal condition, gene expression was visibly achieved. Since it was observed that the protein remained in the inclusion body formed in the centrifuge after sonication, it was continued with the pellet after centrifugation and examined by SDS-PAGE in the control supernatant. After this process, the protein was obtained purely by various His-tag purification methods. Proteins expected to appear at an average size of 29 kDa were confirmed by SDS-PAGE and Western Blot Analysis. Liposomes are small artificial vesicles of spherical shape that can be formed from cholesterol and non-toxic natural phospholipids. Because of their size and hydrophobic and hydrophilic character (as well as biocompatibility), liposomes are promising systems for drug delivery. Liposomes can mimic the cell membrane. Different lipid compositions can be used to obtain the desired liposome type. DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), an example of Phosphatidylcholines (PC), was used in this study. The proteoliposome consists of liposomes containing aquaporin. For 2.5 mg/ml DOPC lipid, aquaporin with a concentration of 0.5 mg/ml was dissolved in 10 ml of PBS and 0.05M OG detergent was added to obtain a proteoliposome, and aquaporin-free liposome was obtained. Control liposome and proteoliposome were treated with 0.85M NaCl and the water transfer rate of aquaporins was measured with a steady flow light scattering spectrometer device. Here, it was determined that the water passage of liposomes with aquaporin was faster. Aquaporin-bearing proteoliposomes lose water at a much higher rate than control liposomes. This was explained as an indication that aquaporin obtained from the Geobacillus Thermoleovorans ARTR1 strain was working. The identification and characterization of novel aquaporin proteins from both halophilic and thermophilic bacteria within the scope of this project are of great importance in terms of their contribution to the literature.
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ÖgePiezomimetic ceramic production for bone biomaterial development(Graduate School, 2023)Bone is a composite tissue that contains various materials, including proteins, crystalline molecules, ions and cells. This complex structure is under study to gain a better understanding of bone properties, especially for the development of bone-like biomaterials that can be used for various purposes. The composite nature of the bone makes it difficult to mimic all of its properties in bone biomaterials. Bone biomaterials can be used as in-vivo implants or for in-vitro tissue development. Various types of materials, such as polymers, ceramics, metals and biological molecules, are used for these purposes. In addition to the structural properties of bone, it has been observed that the surface characteristics of the bone play a crucial role in the growth and differentiation of certain bone cells. This has opened the area for bone topography mimicking materials. Some studies focus on formation of irregular surfaces through physical or chemical methods, while some other studies focus on direct imprinting of the bone surface topography onto certain materials like polymers. In addition to its physical and chemical properties, bone also has electrical properties, which has significant importance. Bone generates electrical currents and electric fields under mechanical stress. In terms of this property, bone shows the nature of a piezoelectric material. The piezoelectric effect observed in bone is produced by collagen molecules and has very important effects on the metabolism of bone growth. Charged ions, such as calcium, intercellular fluids, cytoskeleton proteins, voltage-dependent channels and macromolecules with electrical charges such as growth factors, all contribute to bone healing as they are influenced by the electrical fields generated in the bone. By mimicking the electrical properties of bone, it is possible to develop more effective bone biomaterials. With advancements in material science, biocompatible piezoelectric materials have been developed to mimic the electrical properties of bone. These piezoelectric materials can produce electrical currents and fields to in response to externally applied mechanical forces. In the light of this information, it is clear that both topography and piezoelectricity play a crucial role in bone tissue engineering. In this thesis, these properties were combined to develop new types of materials. First, a novel method was developed to transfer the topography of bone surfaces onto hard metal surfaces. Using these metals with surfaces as master molds, ceramics were produced to have both bone surface mimicking characteristics and piezoelectric properties. Cortical bones were used as bone templates for the bone mimicking materials production. Bones were decellularized with a developed method utilizing di-sodium tetraborate as a cross-linker, to preserve surface hardness of the bones. To produce bone mimicking metal surfaces, the positive bone surfaces were molded in silicone rubber under pressure and heat. These rubber molds were used for transferring bone surface structure to special wax. The molded wax materials were then used to create a negative copy of bone topography in plaster molds by using heated furnace. In the last step, the plasters were filled with brass metal under vacuum to imprint the negative bone topography onto metal as positive structures. After this step, the metal molds were used as master molds in a hydraulic press for transferring the surface topography of metals onto ball milled KNN (potassium sodium niobate) ceramic powder to form green bodies. The green bodies were sintered under temperature controlled conditions to produce intact ceramic materials. The piezoelectric properties of sintered ceramics were gained under a strong electric field. Stereo light microscopy, AFM (atomic force microscopy), SEM (scanning electron microscopy), XRD (X-Ray diffraction) analysis, charge counting and compression tests were used to analyze the results. The analysis has demonstrated the successful production of piezoelectric KNN ceramics with bone surface topography characteristics. By combining topographic and piezoelectric properties, a new term, «piezotopography» is proposed for these type of materials. «Piezotopography» refers to functional topographic structures which have piezoelectric properties. «Piezotopograhic material» refers to materials that possess both functional surface structures and piezoelectric properties. Additionally, by combining mimetic surface feature and piezoelectric properties, a new term, «piezomimetic» is proposed. «Piezomimetic material» refers to materials having functional mimetic surface which have piezoelectric properties. In summary, a new method has been developed to produce ceramics with both bone surface mimicry and piezoelectricity properties.
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ÖgeDetecting binding activity of a therapeutic monoclonal antibody targeting vascular endothelial growth factor using surface plasmon resonance(Graduate School, 2024-07-25)The use of biopharmaceuticals, particularly monoclonal antibodies (mAbs), has become a major application following advancements in recombinant DNA technology. The treatment of a wide range of diseases, among them cancer, asthma, cardiovascular conditions, infections, inflammatory and autoimmune disorders, including allergies, has become possible with the development of mAbs. Surface plasmon resonance (SPR) is a well-known and reliable method that can evaluate biomolecular interactions in real-time in terms of binding affinity and kinetics without requiring labeling procedures. Using SPR technology to determine the kinetic parameters of a mAb molecule resulting from its binding with its target receptor is a common approach to characterize the binding activity of the final product. To characterize the binding kinetics between two molecules, one molecule is first stabilized on the sensor chip surface by immobilization or capturing steps. Then, the other molecule is flowed over the sensor chip surface at variety concentrations in a running buffer to observe the interaction between the two molecules and to determine the resulting kinetic parameters. In this study, after an IgG monoclonal antibody was captured on a Protein A chip surface, the target VEGF121 growth factor was flowed over the surface at five different concentrations. By employing single-cycle kinetic analysis, the kinetic parameters were measured, providing information about the interaction between the two molecules. To develop a robust kinetic assay, it was necessary to optimize several parameters, including the capture molecule (mAb) concentration, the concentration range of the VEGF121 molecule, dissociation time, regeneration time, and the established method sequence. While evaluating the robustness and reliability of the method, important factors considered included quality control parameters from the evaluation software, plotted residuals, and the consistency of Rmax and KD values between replicates. As a result of the method development studies, a capture concentration of 13.4 nM, a dissociation time of 240 seconds and a regeneration time of 30 seconds were decided upon, ensuring that the deviation in KD values between repeated studies was less than 20 %. After the KD value was obtained with low deviation between replicate studies, qualification studies including specificity, system precision, repeatability and intermediate precision parameters were initiated. As a result of 6 replicate studies conducted by two different analysts, the percentage difference between the analysts was determined to be 0.14%. Additionally, kinetic binding studies were performed with mAb molecules from 12 different production batches to demonstrate the effectiveness of the developed method in determining the binding activity of mAb molecules in different lots. These studies showed quite similar kinetic parameters for different production batches.
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ÖgeInvestigation of the effects of abrb and cody deletions on the bacilysin overproducer B. subtilis HWA strain(Graduate School, 2024-07-12)Bacillus subtilis is a gram-positive, rod-shaped bacterium and a highly studied model organism. It has a highly adaptable metabolism and diverse physiological states regulated according to environmental conditions. B. subtilis species go through sporulation to form endospores that can survive harsh conditions such as high temperatures, and UV radiation. They can also form biofilms, attach to plant roots or fungal hyphae, take up extracellular DNA by its natural competence, show surface motility, produce and secrete secondary metabolites. Approximately 4-5% of the B. subtilis genome codes for secondary metabolites, including antibiotic bacilysin. Bacilysin, the main focus of this study, is a non-ribosomal dipeptide by linking non-proteinogenic amino acid anticapsin and L-alanine. Bacilysin causes selective cell wall disruption against bacteria, fungi, and algae species, some of which are pathogenic. Bacilysin is a pleiotropic signaling molecule for B. subtilis cells as it affects diverse cellular functions such as sporulation, germination and outgrowth. Previous studies have shown that the absence of bacilysin can have negative effects on spore quality and germination. A bacilysin non-producer strain was more sensitive to heat, chemicals and lysozyme. Additionally, comparative transcriptome analysis of B. subtilis PY79 and a bacilysin non-producer strain revealed that some genes related to competence development and biofilm formation are also affected by bacilysin. In B. subtilis, bacilysin biosynthesis relies on the expression of the bacABCDEF operon and a monocistronic gene bacG. Bacilysin production is regulated according to both external and internal factors. It has been established that growth conditions such as medium contents, temperature, and pH affect bacilysin production level. At the transcriptional level, bacilysin biosynthesis is regulated mainly via two mechanisms: quorum sensing pathway and stringent response that occur through the direct-action of the positive transcriptional regulators, including ComA~P, Spo0A~P, and LutR as well as negative transcriptional regulators AbrB, CodY, and ScoC. Bacilysin has broad-range activity against bacteria, with heat stability up to 15 min at 100°C, and activity within the pH range of 1.4 to 12.0. These characteristics give bacilysin significant clinical importance and make it an effective alternative to traditional drugs and biocontrol agents. However, bacilysin is produced at low levels, cannot be extracted with organic solvents, and has a low isolation yield. In our group, the bacilysin production level was increased at 2.87- fold via editing the 5' untranslated region (5'UTR) of the bac operon using the CRISPR/Cas9 approach, thereby obtaining the bacilysin overproducing strain B. subtilis HWA. Subsequently, to further boost the bacilysin production level in the over-producing strain B. subtilis HWA, the aim of this study was to examine how production levels are affected by eliminating the global regulators AbrB and CodY. To achieve this, the mutant strains B. subtilis PY79-GT0A (ΔabrB::cat) and B. subtilis PY79-GT0C (unkU::spc ΔcodY) were constructed by transforming competent PY79 cells with chromosomal DNA from the abrB deleted mutant strain B. subtilis BAL 373 (trpC2 pheA1 ΔabrB::cat) and the codY deleted mutant strain B. subtilis TMH 307 (trpC2 unkU::spc ΔcodY), respectively. Similarly, the mutant strains B. subtilis HWA HWA-GTA (ΔabrB::cat) and B. subtilis HWA-GTC (unkU::spc ΔcodY) were constructed by transforming competent HWA cells. Furthermore, the codY-abrB double mutant strains B. subtilis PY79-GT0AC (ΔabrB::cat unkU::spc ΔcodY) and B. subtilis HWA HWA-GTAC (ΔabrB::cat unkU::spc ΔcodY) were constructed by transforming competent cells of GT0A and GTA with chromosomal DNA from B. subtilis TMH 307. Potential tryptophan and/or phenylalanine auxotrophic mutants were eliminated by restreaking on solid Spizizen Minimal Media. Bacilysin phenotypes of the selected mutants were first detected by transferring colonies via toothpicks onto bioassay plates using Staphylococcus aureus ATCC 9144. Subsequently, mutants were grown in PA media for 16-18 hours and the bacilysin levels in their culture fluids were detected via paper disk-diffusion bioassay. Results showed that abrB disruption in HWA and PY79 cells significantly increased bacilysin production, though each strain was affected differently based on its baseline bacilysin production level. The bacilysin level in HWA-GTA increased by 7.7% relative to parental HWA strain, while PY79-GT0A displayed a 21.8% bacilysin level relative to its parent PY79. Interestingly, while codY mutation alone did not significantly affect bacilysin activity in HWA or PY79, adding codY mutation to AbrB mutants of both strains caused further increases of 22.1% and 13.4% relative to HWA and HWA-GTA, respectively, and 25.7% and 3.2% relative to PY79 and PY79-GT0A, respectively. In a final attempt to further enhance bacilysin production, the scoC mutation as an additional negative regulator of the bac operon was combined with the abrB-codY double mutation strain. However, the constructed triple mutant strain could not grow in liquid media, demonstrating that simultaneous disruption of these three global regulators severely compromised growth abilities of B. subtilis cells. In summary, the findings of this thesis study are important to revealing that concurrent inactivation of AbrB and CodY, two key negative regulators of bacilysin biosynthesis, provides the potential for improving bacilysin production levels further, even in the high-producing strain HWA.