Transcript Figure 1. Schematic illustration of principal of single molecule

Food Nanotechnology Lab
Kyung Hee Univ. Food Science and Biotechnology
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
Figure. 1 Scheme of systhesis process
and preparation of PLGA-PEG-COOH diblock copolymer by conjugation. Acid
terminated PLGA was conjugated to a
heterofunctional PEG, NH₂-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 emulsionevaporation method. Oleic acid coated iron oxide
nanoparticle (dia. 10-40 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.
Comparison of PLGA-PEG-COOH and PLGA magnetic particles
Development of next generation DNA
sequencer based on solid state nanopore
Min-Cheol Lim Kwan-Hyung Lee
(이관형)
(임민철)
▪ 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
Principal of single molecule detection using nanopore
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.
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 (handpainting) > lipid bilayer > P-PDMS
Increase the dwell time of DNA translocation by ionic liquids
Figure. 3 Microscope and SEM image of PLGA-PEGCOOH (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.
Figure. 4 Microscope image
of GFP expressed E. coli
O157:H7 with PLGA-PEGCOOH magnetic particle.
Mucin functionalized polydimethylsiloxane
(PDMS) microstructure: A novel tool for
bacterial adhesion test in vitro
Surface topography of biomimetic microstructure
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 ㎛.
Bacterial surface coverage on microstructure
B
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 AC are 100 ㎛.
C
At 500 mV
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.
Min-Cheol Lim
(임민철)
▪ Imitate human intestinal villi structure and physicochemical
parameter to replace in vivo bacterial adhesion test
▪ Elucidate the mechanism of bacterial adhesion vs surface characteristics
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).
A
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.
Ji-Hoon Jung
(정지훈)
Negative deflection and wrinkle formation by plasma treatment
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.
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
Kyung Hee niversity
Food Nanotechnology Lab
Kyung Hee Univ. Food Science and Biotechnology
Inactivation of virulence related wabG gene from
2,3-Butandiol producing Klebsiella pneumoniae
and Klebsiella oxytoca
Jun-Ho Jang (장준호)
▪ 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.
Strategy for the disruption of wabG gene of Klebsiella strains.
The role of lipopolysaccaride and capsular
polysaccharide of Klebsiella spp. on adhesion
and invasion to human epithelial cell
Duyen (유인)
▪ 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.
Klebsiella spp express GFP
Invasion of Klebseilla spp. in human epithelial cell
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.
Characteristic of wabG mutant strains
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.
Figure 3. FE-SEM analysis of the surface morphology of K.
pneumoniae 2242 (A), K. pneumoniae 2242ᐃwabG (B), K.
oxytoca 1686 (C), K. oxytoca 1686ᐃwabG (D), K. oxytoca
43863 (E), and K. oxytoca 43863ᐃwabG (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
s h o w e d d i s t i n c t i v e c e l l t o c e l l
boundaries. Scale bar is 1 μm.
Figure 4. Visualization of the capsules expression in K.
pneumoniae 2242 (A), K. pneumoniae 2242ᐃwabG (B), K.
oxytoca 1686 (C), K. oxytoca 1686ᐃwabG (D), K. oxytoca
43863 (E), and K. oxytoca 43863ᐃwabG (F). The capsules
were shown in white layer around bacterial surface.
Electroporation
Results
GFP expression
Figure 1. 1 and 2: K. peumoniae
KCTC
2242
pET28a-gfp
was
incubated at 300C for 30 h; 3 and 4:
K. oxytoca ATCC 43863 pET28a-gfp
was incubated at 300C for 20 h and
30 h; 5 and 6: K. oxytoca KCTC 1686
pUC18-gfp was incubated at 300C 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
43863 (1,3 and 5: wabG mutant type; 2, 4 and 6:
wild type).
Development of Theranostic Agent using
PHA Synthase
▪Make fusion protein for drug delivery using PHA synthase
Mucin Fuctionalized Impedimetric Biosensor
for Monitoring Bacterial Adhesion
▪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
(A)
(B)
A33scFv
Specificity to tumor cell (A33scFv positive cell)
Flexible
Linker
Separating two fusion protein to enhance flexibility1
GFP
Ah-Young Kim (김아영)
Figure. 1 (A) Eqiuvalent circuit; Rs = solution resistance,
Ret = electron transfer resistance, Cdl = double layer
capacitance
Ret 1 , Cdl1: between gold surface and mucin layer
Ret 2 , Cdl2: 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; V3Studio (Princeton Applied Research).
Hee Su Kwon(권희수)
PHA
synthase
Exhibiting green fluorescence (Diagnosis)
Making particle possible to encapsulating drug
(Therapy)
▪ 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
Figure 1. A modified PHA synthase forms micelles with its PHA polymer chain (A). FESEM image of drug loaded PHA particles
In vitro targeting of A33scFv fused PHB particles and competition assay
Wild type K. pneumoniae KCTC 2242 on mucin surface
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 A33scFvGFP 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.
Verification of drug loading capability using model molecule
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. (R2= 0.99)
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.
Kyung Hee University