Presentation on theme: "K y u n g H e e n i v e r s i t y Food Nanotechnology Lab Kyung Hee Univ. Food Science and Biotechnology Development of next generation DNA sequencer based."— Presentation transcript:
K y u n g H e e n i v e r s i t y Food Nanotechnology Lab Kyung Hee Univ. Food Science and Biotechnology Development of next generation DNA sequencer based on solid state nanopore Min-Cheol Lim ( 임민철 ) Figure 1. Schematic illustration of principal of single molecule detection by nanopore. Charged molecule such as DNA can migrate through nanopore by applied bias voltage. That will temporarily block the ionic current level. The magnitude and duration of this current blockade can be used to elucidate the structure of molecule. Principal of single molecule detection using nanopore Reduce the noise of solid state nanopore chip by organic film Figure 2. Power density spectrum of individual solid chip treated with different organic layer compared with lipid bilayer. Noise level : bare chip > SU-8 > PDMS (hand- painting) > lipid bilayer > P-PDMS Increase the dwell time of DNA translocation by ionic liquids Figure 3. 1 M EMIM-Cl (1-ethyl-3-methylimidazolium chloride) was used for electrolyte to monitoring the translocation of DNA. Λ-DNA (48.5 kbp) was used as target molecule. At 500 mV Young-Rok Kim ( 김영록 ) Professor/Principal Investigator Food Nanotechnology Lab Dept. of Food Science and Biotechnology Kyung Hee University Capturing and concentration of Escherichia coli O157:H7 from food sample using PLGA-PEG immunomagnetic micro-particle Kwan-Hyung Lee ( 이관형 ) Development of an immunogenic capturing system for the detection Escherichia coli O157:H7 from food sample Improvement of the capturing ability of immunomagnetic particle toward target bacteria by controlling the surface morphology Mucin functionalized polydimethylsiloxane (PDMS) microstructure: A novel tool for bacterial adhesion test in vitro Ji-Hoon Jung ( 정지훈 ) Fabrication of biomimetic microstructure Figure 1. Schematic illustration of fabrication of biomimetic microstructure. negative pressure formed by evaporation provide drive force for surface strain (force direction was descripted in the red box). Figure 2. Surface topography generated on the PDMS surface. (A) optical microscopy image of whole microstructure generated on the PDMS (B) AFM (atomic force microscopy) image of crack and surface crease pattern induced by extremely high compressive stress. (C) Ordered wrinkle induced by tolerable compressive stress. (a-c) SEM images of wrinkle pattern on the PDMS surface at the position of figure 4A. Scale bar in a-c are 20 ㎛. Figure 3. Transmission microscopy image of K. pneumonia 2242 (A) adherent on biomimetic microstructure. (BMMS) (B) ordered wrinkle surface. (C) flat surface. Scale bar in A- C are 100 ㎛. Min-Cheol Lim ( 임민철 ) Kwan-Hyung Lee ( 이관형 ) ▪ Imitate human intestinal villi structure and physicochemical parameter to replace in vivo bacterial adhesion test ▪ Elucidate the mechanism of bacterial adhesion vs surface characteristics Figure 4. Schematic illustration of production of wrinkle pattern by negative deflection to the droplet. PDMS wrinkle is formed by plasma treatment. The principle of wrinkle formation is shown in the red box. Negative deflection and wrinkle formation by plasma treatment Figure. 4 Formation of nanopore in PDMS membrane using microneedle. At the moment of micro-pore developing, the PDMS around micro-pore is recovered to the center. Because of this process, the diameter of micro-pore is decreased. Surface topography of biomimetic microstructure Bacterial surface coverage on microstructure AB C ▪ Enhance the signal to noise ratio by reducing the inherent noise of solid nanopore chip ▪ Increase the dwell time of DNA translocation through nanopore Establish the noble ▪ method for nanopore fabrication Bacterial growth and motility in the confined space and microchannels Figure 5. (A and B) Transmission microscopy images of bacteria, A ; E. coli O157:H7 (gfp) and B ; B. cereus, sandwiched between PDMS wrinkle and slide glass. (C) Fluorescence microscopy image of E. coli O157:H7 was taken from the same region of Figure 5A. (D) AFM surface scan of the periodic wrinkle patterns of PDMS substrate. Z-scale is 1 μm. (a-c) The sequential images of E. coli O157:H7 moving along the microchanne Figure. 1 Scheme of systhesis process and preparation of PLGA-PEG-COOH di- block copolymer by conjugation. Acid terminated PLGA was conjugated to a heterofunctional PEG, NH ₂ -PEG-COOH, utilizing standard EDC/NHS mediated chemistry. PLGA was reacted with EDC and NHS in an organic solvent at room temperature. Preparation of antibody coated PLGA-PEG-COOH magnetic particle Figure. 2 Scheme of PLGA-PEG-COOH magnetic particle. The particle was prepared by emulsion- evaporation method. Oleic acid coated iron oxide nanoparticle (dia nm) was added into dichloromethane with PLGA-PEG-COOH. While the solvent was evporated, iron oxide nanoparticle held inside of particles. And, 0.1 ㎎ of particles is overlaied with 2 ㎍ of anti-E. coli O157 monoclonal antibody using EDC/sulfo-NHS chemistry. Figure. 3 Microscope and SEM image of PLGA-PEG- COOH (a) and PLGA (b) magnetic particle (scale bar = 10 ㎛ ). Using the common solvent emulsion-evaporation method, this experiment has shown that replacing hydrophobic polylactic-glycolic-acid (PLGA) by amphiphilic poly-lactic-glycolic-acid-poly-ethylene-glycol (PLGA-PEG) led to spiky microparticle. Amphiphilic copolymers attribute these structural changes to interfacial instabilities at the emulsion droplet interface during solvent evaporation. Comparison of PLGA-PEG-COOH and PLGA magnetic particles Figure. 4 Microscope image of GFP expressed E. coli O157:H7 with PLGA-PEG- COOH magnetic particle.
K y u n g H e e U n i v e r s i t y The role of lipopolysaccaride and capsular polysaccharide of Klebsiella spp. on adhesion and invasion to human epithelial cell Duyen ( 유인 ) Figure 2. Deletion of chloramphenicol gene in mutant Klebsiella strains. Mutant Klebsiella strains was transformed with 707-FLPe plasmid expressing FLP. After FLP expression, one part of mutant Klebsiella colony was incubated in no chloramphenicol LB Media and the other part was incubated in chloramphenicol LB media. Mutant Klebsiella strains that don’t grow in chloramphenicol LB media was chosen and confirmed deletion of chloramphenicol gene by genomic DNA PCR. Strategy for the disruption of wabG gene of Klebsiella strains. Figure 1. wabG gene knock out process. Klebsiella strains was transformed with pRedET. After expression of Red α, Red β Red γ, Klebsiella strains and WFCFW cassette were transformed. Klebsiella strains that grown in chloramphenicol plate was chosen and confirmed wabG mutation by genomic DNA PCR. Inactivation of virulence related wabG gene from 2,3-Butandiol producing Klebsiella pneumoniae and Klebsiella oxytoca Jun-Ho Jang ( 장준호 ) Characteristic of wabG mutant strains Figure 3. FE-SEM analysis of the surface morphology of K. pneumoniae 2242 (A), K. pneumoniae 2242wabG (B), K. oxytoca 1686 (C), K. oxytoca 1686wabG (D), K. oxytoca (E), and K. oxytoca 43863wabG (F). The surfaces of wild type Klebsiella species were shown to be covered with a thick layer of capsular polysaccharide. On the other hand, wabG mutant strains were absent of such layer and thus showed distinctive cell to cell boundaries. Scale bar is 1 μm. Figure 4. Visualization of the capsules expression in K. pneumoniae 2242 (A), K. pneumoniae 2242wabG (B), K. oxytoca 1686 (C), K. oxytoca 1686wabG (D), K. oxytoca (E), and K. oxytoca 43863wabG (F). The capsules were shown in white layer around bacterial surface. Mucin Fuctionalized Impedimetric Biosensor for Monitoring Bacterial Adhesion Ah-Young Kim ( 김아영 ) Wild type K. pneumoniae KCTC 2242 on mucin surface Klebsiella spp express GFP GFP expression Electroporation Invasion of Klebseilla spp. in human epithelial cell Results Figure 1. 1 and 2: K. peumoniae KCTC 2242 pET28a-gfp was incubated at 30 0 C for 30 h; 3 and 4: K. oxytoca ATCC pET28a-gfp was incubated at 30 0 C for 20 h and 30 h; 5 and 6: K. oxytoca KCTC 1686 pUC18-gfp was incubated at 30 0 C for 30 h (1,3 and 5: wabG mutant type; 2, 4 and 6: wild type) Figure 2. 1, 2: K. pneumoniae KCTC 2242; 3, 4: K. oxytoca KCTC 1686; 5, 6: K. oxytoca ATCC (1,3 and 5: wabG mutant type; 2, 4 and 6: wild type). Development of Theranostic Agent using PHA Synthase Hee Su Kwon( 권희수 ) In vitro targeting of A33scFv fused PHB particles and competition assay Verification of drug loading capability using model molecule Figure 1. A modified PHA synthase forms micelles with its PHA polymer chain (A). FE- SEM image of drug loaded PHA particles Figure 2. A) Optical and fluorescence images of HT29 (A33negative) (a and b) and SW1222 (A33positive) (c and d) colon cancer cells after treatment with A33scFv-GFP fused PHB particles. Cells were treated with PHA nanoparticles produced by A33scFv- GFP fused PHA synthase. Scale bar is 30 µm. B) Competition assay results of A33scFv and A33scFv-GFP fused PHA synthase. The fluorescence intensity was decreased as A33scFv concentration increased. Figure 3. A. Hydrophilic drug loading capability. A hydrophilic dye, Fluorescein sodium salt(FSS) was employed to study hydrophilic drug loading capacity. B. Lipophillic drug loading capability. A lipophilic dye, nile red was employed to study lipophilic drug loading capacity. Scale bar is 10 µm. ▪ Make fusion protein for drug delivery using PHA synthase ▪ Confirmation of function of fusion protein → A33scFv: binding ability, GFP: fluorescence ability, PHA synthase: particle synthesis ▪ Verification of drug loading capability using model molecules. ▪ Evaluating drug release profile and the efficacy of the delivery system A33scFvSpecificity to tumor cell (A33scFv positive cell) Flexible Linker Separating two fusion protein to enhance flexibility 1 GFPExhibiting green fluorescence (Diagnosis) PHA synthase Making particle possible to encapsulating drug (Therapy) ▪ Elimination of LPS that is major virulence factor of Klebsiella spp. by mutation of wabG gene which plays a key role in synthesis of outer core LPS. ▪ Observing characteristic of wabG mutant strains. ▪ Confirming glucose consumption (G.C) and 2,3-BDO production of wabG mutant strains. ▪ Development of an impedimetric sensing system to measure the binding characteristics of Klebsiella peumoniae KCTC 2242 to models surface ▪ Examine the role of outer core of LPS and capsule on the adhesion of Klebsiella peumoniae KCTC 2242 to an epithelial cell-like surface Equipment Figure. 1 (A) Eqiuvalent circuit; R s = solution resistance, R et = electron transfer resistance, C dl = double layer capacitance R et 1, C dl 1: between gold surface and mucin layer R et 2, C dl 2: between mucin layer and solution We measured impedance between mucin layer and solution. (B) Equipment for electrochemical detection using fluidic chamber ; Fluidic camber consists of platinum electrode, jig-body, inlet & outlet and screw type holder. Samples were injected at a constant rate by syringe pump. The data was obtained from an electrochemical analyzer VersaSTAT3 (Princeton Applied Research, Tennessee, USA). Analysis of electrochemical signal using computer software; V3- Studio (Princeton Applied Research). (A) (B) Figure. 2 (A) Surface functionalization confirmed through impedimetric analysis of gold surface during surface functionalization. (a) Bare gold (b) 11-mercapto-1-undecanol (c) Epichlorohydrin (d) Mucin (e) Bacteria. Impedance value was increased by processing with surface modification. (B) Bode plots of K. pneumoniae KCTC 2242 in mucin functionalized gold surface. (C) Normalized impedance change(NIC) at 0.1 Hz. NIC(%) levels were increased concentration dependant manner. (R 2 = 0.99) ▪ We present the role of LPS of Klebsiella species when they invade human epithelial cell. By using plasmid-harboring gene for GFP, we can monitor the presence or invasion of Klebsiella species in vivo as well as their intensity instead of colony counting on agar plate. This method will give a quick and accurate means of monitoring the invasion process of pathogenic bacteria. In this study we evaluated the role of outer core LPS in invasion to human epithelial cells. Food Nanotechnology Lab Kyung Hee Univ. Food Science and Biotechnology