CELL AND PROTEIN-BASED SENSING SYSTEMS FOR THE DETECTION OF ENVIRONMENTALLY AND PHYSIOLOGICALLY RELEVANT MOLECULES

The detection of small molecules in complex sample matrices such as environmental (surface and ground water, sediment, etc.) and biological (blood, serum, plasma, etc.) samples is of paramount importance for monitoring the distribution of environmental pollutants and their patterns of exposure within the population as well as diagnosing and managing diseases. Biosensors have demonstrated a singular ability to sensitively and selectively detect analytes in complex samples without the need for extensive sample preparation and pretreatment. Nature has demonstrated myriad examples of exquisite selectivity in spite of complexity and we seek to take advantage of that attribute in the development of novel biosensing systems. In the work presented here, we have developed both cell- and proteinbased biosensing systems for the detection of hydroxylated polychlorinated biphenyls (OH-PCBs) and protein-based sensing systems for the detection of glucose. In the development of a whole-cell sensing system, the regulatory protein, HbpR, and its associated promoter was used to modulate the expression of luciferase. Additionally, the effector binding domain of HbpR, HbpR-A, was isolated and modified with a solvatochromic fluorophore resulting in a proteinbased sensing system. For the detection of glucose, two different glucose binding proteins were engineered in an effort to tailor their characteristics, such as binding affinity and thermal stability, to develop a rugged, sensitive proteinbased sensing system. We envision that these biosensing systems will find applications in the areas of environmental pollutant monitoring and the management and treatment of diseases such as diabetes

인간 미각 수용체 및 광수용체의 기능적 생산, 분석 및 응용

학위논문(박사) -- 서울대학교대학원 : 공과대학 협동과정 바이오엔지니어링전공, 2023. 2. 박태현.G 단백질 결합 수용체(GPCR)는 가장 큰 부류의 막횡단 단백질 패밀리로 시각, 미각, 후각, 신경전달물질 및 호르몬과 같은 대부분의 세포 반응을 매개한다. GPCR의 활동과 기능을 측정하는 것은 식품, 환경 및 생물의학 응용 분야에서 매우 중요하다. 따라서 기능적인 GPCR을 생산하고 공정하는 것은 GPCR 연구 및 실제 응용을 위한 도구를 개발하는 데 중요하다. 인간에게는 시각, 미각, 후각, 청각, 촉각의 다섯 가지 감각이 있다. 오감 중 시각, 미각, 후각은 GPCR 신호전달을 포함한다. A 클래스 GPCR에는 시각과 후각을 담당하는 광수용체와 후각 수용체가 포함된다. 클래스 C GPCR에는 종종 "맛있는" 맛으로 알려진 단맛과 감칠맛에 대한 미각 수용체가 포함된다. 본 연구에서는 다양한 접근 방식을 수행하여 인간의 미각 수용체와 시각 광수용체를 생산하였다. 인간의 단맛 및 감칠맛 수용체를 활용하기 위해 대장균에서 과발현하기 어려운 전체 수용체를 사용하는 대안으로 수용체의 주요 리간드 결합 도메인을 대장균으로부터 대량생산하였다. 생산된 T1R2 VFT와 T1R1 VFT는 실용적인 방식으로 단맛과 감칠맛을 평가하기 위해 트립토판 형광 분석으로 독립적으로 분석되었고 전도성 나노 물질과 결합하여 단맛 및 감칠맛 센서를 제작하였다. 대장균에서 생산한 인간 광수용체 나노디스크와 동물세포에서 생산한 광수용체 발현 바이러스는 여러 분석과 응용을 통해 인간 광수용체의 생리학적 및 생화학적 메커니즘을 더 잘 이해하고 이를 통한 시각 복원 기술 개발에 적용하고자 하였다. 첫째, 인간의 단맛 및 감칠맛 수용체의 주요 리간드 결합 도메인인 T1R2 VFT 및 T1R1 VFT는 전체 수용체를 사용하는 대안 전략으로 대장균에서 기능적으로 생성하였다. VFT의 기능은 각각 대표적인 맛 물질인 수크로스와 MSG를 사용하여 트립토판 형광 분석을 사용하여 테스트하였다. 기능적으로 생산된 VFT는 단맛과 감칠맛 물질을 감지하기 위한 생체 재료로 사용하기 위해 분석 및 검증하였다. 둘째, 인간의 단맛 수용체의 주요 리간드 결합 도메인을 이용하여 실질적인 단맛 물질을 평가하였다. 인간 단맛 수용체의 T1R2 VFT를 사용하여 단맛을 측정하는 비교적 간단한 접근법인 트립토판 형광 분석을 사용하여 단맛 물질에 대한 효능을 평가하였다. 트립토판 분석 결과는 수용체 수준에서 리간드 결합을 해석하는 빠르고 간단한 방법을 제공하기 위해 수크로스에 대한 상대적인 단맛으로 표현되었다. 또한, T1R2 VFT를 CNT-FET의 전극에 고정화하여 단맛을 감지하는 실용적인 단백질 기반 바이오 센서를 제작하였다. 셋째, 인간 감칠맛 수용체의 주요 리간드 결합 도메인을 사용하여 감칠맛 물질과 물질간의 시너지 효과를 트립토판 형광 분석을 통해 평가하였다. 또한, T1R1 VFT는 플로팅 전극 기반 CNT-FET에 고정하였고 전도성 고분자 하이드로겔과 결합하였다. T1R1 VFT를 포함하는 하이드로겔 매개 CNT-FET는 식품에서 감칠맛 화합물을 검출하는 데 유용한 도구일 뿐만 아니라 식품 부문 및 기초 연구에서 중요한 응용을 가질 것으로 예상된다. 마지막으로 광수용체는 대장균과 동물 세포에서 생산하여 다양한 실험과 응용에 사용하였다. 대장균에서 생산된 광수용체 나노디스크는 광학 스위칭과 같은 잠재적인 광학 응용 분야에 사용하기 위한 특성을 측정하는 데 사용하였다. 동물세포에서 생산된 광수용체 발현 바이러스는 눈에서 뇌로 가는 신경 회로의 시험관 내 모델 개발에 사용하였다. 본 연구에서는 인간의 미각 수용체와 시각 광수용체를 생산하고 공정하는 다양한 접근 방식을 적용하였다. 인간의 미각과 시각에 대한 더 나은 이해와 미래의 식품, 환경 및 생물 의학 응용을 위해 인공적으로 생산한 인간 감각 수용체를 사용한 다양한 분석 및 응용도 조사하였다.G protein-coupled receptors (GPCRs) are the largest transmembrane protein family, mediating the majority of cellular responses such as vision, taste, olfaction, neurotransmitters, and hormones. Measuring the activity and function of GPCR is critical in food, environmental, and biomedical applications. Therefore, producing and engineering functional GPCRs are important in developing tools for GPCR research and practical applications. Humans have five senses: sight, taste, smell, hearing, and touch. Among the five senses, the senses of vision, taste, and smell involve GPCR signaling. Class A GPCRs include photoreceptors and olfactory receptors responsible for vision and smell. Class C GPCRs include taste receptors for sweet and umami taste, often known as palatable taste. In these theses, various approaches to engineer and produce human taste receptors and visual photoreceptors were performed. For utilizing human sweet and umami taste receptors, a primary ligand binding domain of the receptor was mass-produced from E. coli as an alternative strategy to using a whole receptor, which is difficult to overexpress in E. coli. These were analyzed independently with tryptophan fluorescence assay and combined with conducting nanomaterials to evaluate sweet and umami taste in a practical manner. Human photoreceptor NDs produced from E. coli and photoreceptor-expressing virus produced from mammalian cells were analyzed to better understand the physiological and biochemical mechanisms in human photoreceptors and to further apply this knowledge to develop vision restoration technologies. First, a primary ligand-binding domain of a human sweet and umami taste receptor, the T1R2 VFT and T1R1 VFT, were functionally produced from E. coli as an alternative strategy to using a whole receptor. The functions were analyzed by tryptophan fluorescence assay using the representative taste molecule, sucrose, and MSG, respectively. Functionally refolded VFTs have been validated for use as a biomaterial for detecting sweet and umami substances. Second, the ligand-binding domain of the human sweet taste receptor was used to evaluate sweet taste substances in a practical manner. Tryptophan fluorescence analysis, a relatively simple approach for measuring sweet taste using T1R2 VFT of human sweet receptors, was used to assess the efficacy of sweet taste compounds. The tryptophan assay results were expressed as relative sweetness to provide a quick and simple way to interpret ligand binding at the receptor level. Furthermore, a T1R2 VFT was immobilized on the floating electrode of a CNT-FET to develop a protein-based biosensor for the practical detection of sweet compounds. Third, the ligand-binding domain of the human umami taste receptor was used to evaluate umami taste substances and their synergistic effects using tryptophan fluorescence assays. Furthermore, a T1R1 VFT was hybridized with floating electrode-based CNT-FET and combined with a conductive polymer hydrogel. The hydrogel-mediated CNT-FET with T1R1 VFT was anticipated to be a useful tool for detecting umami compounds in foods, as well as a significant application in the food sector and fundamental research. Lastly, the photoreceptors were either produced in E. coli and mammalian cells, and they were used in a variety of experiments and applications. Photoreceptor NDs produced from E. coli were used for measuring the optical properties of their potential optical applications, such as optical switching. Photoreceptor-expressing virus produced from mammalian cells was used for developing in vitro model of a neural circuit for the eye to the brain. In this study, various approaches to engineering and producing human taste receptors and visual photoreceptors were used. Various analyses and applications using engineered human sensory receptors were also investigated for a better understanding of human taste and vision and future food, environmental, and biomedical applications.Chapter 1 Research background and objective 13 Research background and objective 14 Chapter 2 Literature review 17 2.1 G protein-coupled receptors 18 2.2 Sensory transduction in taste and vision 19 2.2.1 Taste signal transduction 19 2.2.2 Visual phototransduction 20 2.3 GPCRs produced from heterologous expression system 22 2.3.1 GPCRs production in prokaryotic cells 22 2.3.2 GPCRs production in eukaryotic cells 23 2.4 Nanobiosensors using natural receptors 24 2.4.1 Protein-based biosensors 25 2.4.2 Nanodisc-based biosensors 25 2.4.3 Nanovesicle-based biosensors 26 2.4.4 Peptide-based biosensors 27 Chapter 3 Experimental procedures 28 3.1 Gene cloning 29 3.1.1 Cloning of T1R2 VFT, T1R1 VFT and photoreceptor genes into E. coli expression vector 29 3.1.2 Cloning of photoreceptor gene into mammalian expression vector 29 3.1.3 Cloning of photoreceptor gene into lentiviral expression vector 30 3.2 Protein production from E. coli 31 3.2.1 Expression and purification of T1R2 VFT, T1R1 VFT and photoreceptors in E. coli 31 3.2.2 Expression and purification of membrane scaffold protein in E. coli 32 3.2.3 SDS-PAGE analysis 33 3.3 Expression of photoreceptors in mammalian cells and construction of HEK-293 stable cell lines expressing photoreceptors 35 3.3.1 Expression of photoreceptors in HEK-293 and R28 cell 35 3.3.2 Lentiviral production and transduction of primary neuronal cells expressing photoreceptors 35 3.3.3 Construction of HEK-293 stable cell lines expressing photoreceptors 36 3.3.4 Calcium imaging analysis 36 3.4 Functional reconstitution 38 3.4.1 Refolding of T1R2 VFT and T1R1 VFT 38 3.4.2 Assembly of photoreceptor NDs 38 3.4.3 Tryptophan fluorescence quenching assay 39 3.4.4 Dynamic light scattering and scan electron microscopy imaging 39 3.5 Fabrication of ligand binding domain of human taste receptors immobilized on CNT-FET sensors 41 3.5.1 T1R2 VFT immobilization on CNT-FET 41 3.5.2 T1R1 VFT immobilization on CNT-FET and formation of hydrogel layers on T1R1 VFT-based sensors 41 3.6 Fabrication of PDMS and photoreceptor ND-filled waveguides 43 3.6.1 Refractive index measurement of photoreceptor NDs 43 3.6.2 Fabrication of photoreceptor ND-filled PDMS-based waveguides and analysis of beam profile intensity 44 Chapter 4 Production of ligand binding domain of human sweet and umami taste receptors 45 4.1 Introduction 46 4.2 Expression and purification of ligand binding domain of human sweet and umami taste receptors in E. coli system 47 4.3 Functional characterization of ligand binding domain of human sweet and umami taste receptors 49 Figure 4.4 Functional assay of T1R1 VFT to MSG using tryptophan fluorescence quenching assay 50 4.4 Conclusions 51 Chapter 5 Ligand binding domain of human sweet taste receptor: Analyses and applications to measure sweet taste 52 5.1 Introduction 53 5.2 Taste ligand efficacy analysis in ligand binding domain of human sweet taste receptor using a tryptophan fluorescence assay 55 5.3 Fabrication and characterization of T1R2 VFT-based CNT-FET 60 5.4 Detection of sweet tastants using T1R2 VFT-based CNT-FET 62 5.5 Measurements of real beverage samples using T1R2 VFT-based CNT-FET 66 5.6 Conclusions 69 Chapter 6 Ligand binding domain of human umami taste receptor: Analyses and applications to measure umami taste 70 6.1 Introduction 71 6.2 Taste ligand efficacy analysis in ligand binding domain of human umami taste receptor using a tryptophan fluorescence assay 74 6.3 Fabrication of hydrogel-mediated CNT-FET using T1R1 VFT 76 6.4 Real-time and selective responses to umami tastants 78 6.5 Measurements of fish extract samples using hydrogel-mediated CNT-FET using T1R1 VFT 82 6.6 Conclusions 86 Chapter 7 Production and functional reconstitution of human photoreceptors 87 7.1 Introduction 88 7.2 Expression and purification of photoreceptors in E. coli system 90 7.3 Structural and functional characterization of photoreceptor NDs 92 7.4 Expression and production of human photoreceptors in mammalian cell 95 7.5 Conclusions 99 Chapter 8 Human photoreceptors: Analyses and applications to measure optical properties 100 8.1 Introduction 101 8.2 Analyses and applications to measure optical properties using photoreceptors produced in E. coli 103 8.2.1 Functional characterization of photoreceptor NDs using tryptophan fluorescence assay 103 8.2.2 Refractive index measurement of photoreceptor NDs 105 8.2.3 Fabrication of PDMS and photoreceptor ND-filled waveguides 109 8.2.4 Waveguiding of photoreceptor NDs using PDMS and photoreceptor ND-filled waveguide 111 8.3 Analyses and applications to measure optical properties using photoreceptors produced in mammalian cell 114 8.3.1 Functionality analysis of photoreceptors using calcium assay 114 8.3.2 In vitro neural circuit using photoreceptor-expressing neural spheroid 116 8.4 Conclusions 119 Chapter 9 Overall discussion and further suggestions 121 Overall discussion and further suggestions 122 Bibliography 127 국문초록 136박

DEVELOPMENT OF LUMINESCENT SENSING SYSTEMS WITH CLINICAL APPLICATIONS

As the move towards the miniaturization of many diagnostic and detection systems continues, the need for increasingly versatile yet sensitive labels for use in these systems also grows. Luminescent reporters provide us with a solution to many of the issues at hand through their unique and favorable characteristics. Bioluminescent proteins offer detection at extremely low concentrations and no interference from physiological fluids leading to excellent detection limits, while the vast number of fluorescent proteins and molecules available allows the opportunity to select a tailored reporter for a specific task. Both provide relatively simply instrumentation requirements and have exhibited great promise with many of the miniaturized systems such as lab-on-a-chip and lab-on-a-CD designs. Herein, we describe the novel employment of luminescent reporters for four distinct purposes. First off, by combining both time and wavelength resolution we have expanded the multiplexing capabilities of the photoprotein aequorin beyond duel-analytes, demonstrating the ability to simultaneously detect three separate analytes. Three semi-synthetic aequorin proteins were genetically conjugated to three pro-inflammatory cytokines (interleukins 1, 6, and 8) resulting in aequorin labeled cytokines with differing emission maxima and half lives to allow for the simultaneous detection of all three in a single solution through the elevated physiological concentration range. Secondly a semi-synthetic aequorin variant has been genetically enhanced to serve as an immunolabel and exhibited the ability to sensitively detect the acute myeloid leukemia marker, CD33, down to the attomole level in addition to improving aequorin imaging capabilities. In the third example, the aequorin complex was rationally, genetically split into two parts and attached to the termini of the cAMP selective cAMP receptor protein (CRP) creating a genetically fused molecular switch. The conformational change experienced by CRP upon the binding of cAMP translates into a loss of bioluminescent signal from aequorin and has shown the ability to respond linearly to cAMP over several orders of magnitude. Lastly, through custom design, a reagentless, portable, fluorescent fiber optic detection system has been developed, capable of being integrated into the body through a heart catheter. The system was able to respond to changes in potassium concentration selectively, reproducibly and reversibly with a fast response time of one minute

Selection and Characterization of Synthetic Antibodies Against Human Rad51

Chemotherapy is the predominant approach for treating cancer. However, disease relapse frequently occurs because chemotherapy often fails to eliminate all tumor cells due to intrinsic or acquired drug resistance. The failure of conventional chemotherapeutics regimes for cancer highlights the need for novel therapeutic interventions. Recent studies have shown that human Rad51, an evolutionarily conserved DNA recombinase required for homologous recombination, exhibits elevated expression in many cancer cells and is implicated in drug resistance after chemotherapy. Targeted inhibition of human Rad51 has been explored as a way to sensitize cancer cells to chemotherapy. Given the properties of antibodies and their fragments as high-affinity inhibitors, we used antibody phage display to generate antigen-binding fragments (Fabs) against human Rad51. We first isolated human Rad51 specific Fabs by screening a synthetic Fab phage display library against recombinant human Rad51. We isolated a human Rad51 Fab, referred to as Fab F2, which bound human Rad51 with a KD of 8.1 nM. Fab F2 inhibited the DNA binding activity of human Rad51 but did not inhibit human Rad51 ATP hydrolysis activity. We converted Fab F2 into an scFv-Fc fragment (scFv: single-chain variable fragment; Fc: glycosylated crystallizable fragment) for expression in human embryonic kidney 293T cells. Overexpression of scFv-Fc fragment in human embryonic kidney 293T cells increased 4.48-fold more sensitivity to the DNA-damaging agent methyl methanesulfonate in clonogenic survival assays. To enable the delivery of Fab F2 into cells we fused it to a cell membrane import tag (FabItag I2) based on a patent (WO 2014005219 A1) from iProgen Biotech Inc. We labeled FabItag I2 with an 800CW fluorophore and showed that FabItag I2 permeated human embryonic kidney 293T cells using fluorescence microscopy and flow cytometry. FabItag I2 increased the sensitivity of human embryonic kidney 293T cells to methyl methanesulfonate by 2 folds in clonogenic survival assays

Selective Immobilization of Fluorescent Proteins for the Fabrication of Photoactive Materials

The immobilization of fluorescent proteins is a key technology enabling to fabricate a new generation of photoactive materials with potential technological applications. Herein we have exploited superfolder green (sGFP) and red (RFP) fluorescent proteins expressed with different polypeptide tags. We fused these fluorescent proteins to His-tags to immobilize them on graphene 3D hydrogels, and Cys-tags to immobilize them on porous microparticles activated with either epoxy or disulfide groups and with Lys-tags to immobilize them on upconverting nanoparticles functionalized with carboxylic groups. Genetically programming sGFP and RFP with Cys-tag and His-tag, respectively, allowed tuning the protein spatial organization either across the porous structure of two microbeads with different functional groups (agarose-based materials activated with metal chelates and epoxy-methacrylate materials) or across the surface of a single microbead functionalized with both metal-chelates and disulfide groups. By using different polypeptide tags, we can control the attachment chemistry but also the localization of the fluorescent proteins across the material surfaces. The resulting photoactive material formed by His-RFP immobilized on graphene hydrogels has been tested as pH indicator to measure pH changes in the alkaline region, although the immobilized fluorescent protein exhibited a narrower dynamic range to measure pH than the soluble fluorescent protein. Likewise, the immobilization of Lys-sGFP on alginate-coated upconverting nanoparticles enabled the infrared excitation of the fluorescent protein to be used as a green light emitter. These novel photoactive biomaterials open new avenues for innovative technological developments towards the fabrication of biosensors and photonic devices

Development of electrochemical nitrite biosensors using cytochrome c nitrite reductase from Desulfovibrio desulfuricans ATCC 27774

Dissertação apresentada para a obtenção do Grau de Doutor em Química Sustentável pela Universidade Nova de Lisboa, Faculdade de Ciências e TecnologiaREQUIMTE ; Fundação para a Ciência e Tecnologia -(POCI/QUI/58026/2004 and SFRH/BD/28921/2006

Utilization of yeast pheromones and hydrophobin-based surface engineering for novel whole-cell sensor applications

Whole-cell sensors represent an emerging branch in biosensor development since they obviate the need for enzyme/antibody purification and provide the unique opportunity to assess global parameters such as genotoxicity and bioavailability. Yeast species such as Saccharomyces cerevisiae are ideal hosts for whole-cell sensor applications. However, current approaches almost exclusively rely on analyte-induced expression of fluorescent proteins or luciferases that imply issues with light scattering and/or require the supply of additional substrates. In this study, the yeast α-factor mating pheromone, a peptide pheromone involved in cell-cell communication in Saccharomyces cerevisiae, was utilized to create the whole-cell sensor read-out signal, in particular by employing engineered sensor cells that couple the response to a user-defined environmental signal to α-factor secretion. Two novel immunoassays - relying on hydrophobin-based surface engineering - were developed to quantify the α-factor. Hydrophobins are amphiphilic fungal proteins that self-assemble into robust monolayers at hydrophobic surfaces. Two recombinant hydrophobins, either lacking (EAS) or exposing the α-factor pheromone (EAS-α) upon self-assembly, were used to functionalize polystyrene supports. In a first approach (competitive immunoassay), pheromone-specific antibodies initially bound to the functionalized surface (due to the α-factor exposed by the hydrophobin layer) were competitively detached by soluble α-factor. In a second approach, the antibodies were first premixed with pheromone-containing samples and subsequently applied to functionalized surfaces, allowing for the attachment of antibodies that still carried available binding sites (inverse immunoassay). Both immunoassays enabled quantitative assessment of the yeast pheromone in a unique but partially overlapping dynamic range and allowed for facile tuning of the assay sensitivity by adjustment of the EAS-α content of the hydrophobin layer. With a limit of detection of 0.1 nM α-factor, the inverse immunoassay proved to be the most sensitive pheromone quantification assay currently available. Due to the high stability of hydrophobin monolayers, functionalized surfaces could be reused for multiple consecutive measurements. Favorably, both immunoassays proved to be largely robust against the changes in the sample matrix composition, allowing for pheromone quantification in complex sample matrices such as yeast culture supernatants. Hence, these immunoassays could also be applied to study the pheromone secretion of wild-type and engineered Saccharomyces cerevisiae strains. Additionally, a proof-of-concept whole-cell sensor for thiamine was developed by combining the hydrophobin-based immunoassays with engineered sensor cells of Schizosaccharomyces pombe modulating the secretion of the α-factor pheromone in response to thiamine. Since this read-out strategy encompasses intrinsic signal amplification and enables flexible choice of the transducer element, it could contribute to the development of miniaturized, portable whole-cell sensors for on-site application

Exploiting bioluminescence to enhance the analytical performance of whole-cell and cell-free biosensors for environmental and point-of-care applications

The routine health monitoring of living organisms and environment has become one of the major concerns of public interest. Therefore, there has been an increasing demand for fast and easy to perform monitoring technologies. The current available analytical techniques generally offer accurate and precise results; however, they often require clean samples and sophisticated equipment. Thus, they are not suitable for on site, real-time, cost-effective routine monitoring. To this end, biosensors represent suitable analytical alternative tools. Biosensors are analytical devices integrating a biological recognition element (i.e. antibody, receptor, cell) and a transducer able to convert the biological response into an easily measurable analytical signal. These tools can easily quantify an analyte or a class of analytes of interest even in a complex matrix, like clinical or environmental samples, thanks to the specificity of the biological components. Whole-cell biosensors among others offer unique features such as low cost of production and provide comprehensive functional information (i.e. detection of unclassified compounds and synergistic effects, information about the bioavailable concentration). During this PhD, several bioengineered whole-cell biosensors have been developed and optimized for environmental and point-of-care applications. Analytical performance of biosensors have been improved (i.e. low limit of detection, faster response time and wider dynamic range) thanks to synthetic biology and genetic engineering tools. Bacterial, yeast and 3D cell cultures of mammalian cell lines have been tailored at the molecular level to improve robustness and predictivity. Several reporter genes, i.e. colorimetric, fluorescent and bioluminescent proteins, have been also profiled for finding the best candidate for each point-of-need application. Furthermore, spectral resolution of different optical reporter proteins has been exploited and multiplex detection has been achieved. The inclusion of viability control strains provided a suitable tool for assessing non-specific effects on cell viability, correcting the analytical signal and increasing the analytical performance of ready-to-use cartridges

Spatial Regulation of Cell Division by the Min System in E. coli

The E. coli Min system contributes to spatial regulation of cell division by preventing Z ring assembly at cell poles. Critical to our understanding of this spatial regulation by the Min system is the mechanism of action of MinC, an inhibitor of Z ring formation. Even though the Min system has been extensively studied, the molecular mechanism by which MinC antagonizes Z ring assembly is still not very clear, which is the goal of this study. MinC has two functional domains, both of which are able to block cell division in the proper context---MinCn can do so by itself whereas MinCc requires MinD. In this work, we describe the inhibitory mechanism of each domain of MinC on Z ring assembly. First, we show that the septal localization and division inhibitory activity of MinCc/MinD requires the conserved C-terminal tail of FtsZ. Using a genetic screen we identified four mutations in FtsZ that significantly decrease the MinCc/MinD-FtsZ interaction and the toxivity of MinCc/MinD. These mutations are clustered at the conserved C-terminal tail of FtsZ, a region critical for FtsZ-FtsA and FtsZ-ZipA interactions and therefore Z ring assembly. Using this as a clue, we were able to show that the toxicity of MinCc/MinD in blocking division is due to its competition with FtsA and/or ZipA for the tail of FtsZ. In the presence of overexpressed MinCc/MinD, such competition displaces FtsA and/or ZipA from the Z ring to disrupt the integrity and functionality and eventually totally destroy the structure of the Z ring. Second, we studied the interaction between FtsZ and the N terminal domain of MinC. MinCn has been shown to be the anti-FtsZ part of MinC but the detailed mechanism regarding this activity is not known. Previous studies lead to the puzzling observation that MinCn blocks FtsZ polymer sedimentation but does not affect its GTPase. Because the GTPase activity of FtsZ is linked to its polymerization, MinCn is believed to act after the polymerization of FtsZ to shorten FtsZ polymers. Using a similar genetic screen as above, we identified the residues in FtsZ that are critical for the MinCn-FtsZ interaction. These important residues are clustered at the FtsZ dimerization interface, indicating that MinCn attacks FtsZ polymers at the dimer interface. Based on this, a "wedge" model for the action of MinCN on FtsZ is proposed. Collectively, this study encourages us to suggest a more detailed model for how MinC/MinD antagonizes the Z ring formation: MinC/MinD localizes to the Z ring or membrane-associated FtsZ polymers through MinCc/MinD interacting with the conserved C-terminal tail of FtsZ. By directly contacting FtsZ, MinC/MinD prevents Z ring formation in at least two ways: first, MinCc/MinD disrupts the function and structural integrity of the Z ring by interfering with the recruitment of FtsA and/or ZipA; second, this targeting of MinC/MinD to the Z ring brings MinCn in close proximity to FtsZ polymers, which then severs these FtsZ polymers so that the Z ring is completely destroyed. By targeting different regions of FtsZ the two domains of MinC affect different aspects of Z ring formation to achieve synergy in disrupting Z rings. Normally the activity of MinC/MinD is spatially regulated by MinE so that it works only at cell poles to block the formation of any potential polar Z rings. During the course of this study, we discovered another layer of spatial regulation of cytokinesis by MinC/MinD independent of MinE. The accumulated evidence shows that polar Z rings are more sensitive to MinC/MinD than midcell Z rings even in the absence of MinE. In some cases such as in the FtsZ-I374V strain, wild type morphology can be achieved by MinC/MinD without MinE. The mechanism of this differential MinC/MinD sensitivity between polar and midcell Z rings is unknown but it suggests that another layer of spatial regulation of cytokinesis by MinC/MinD exists other than oscillation induced by MinE