Final Exam: May 4th, 9-11am DCL 3211. Last Class: Genetic Engineering 1. Restriction nucleases 2....
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Transcript of Final Exam: May 4th, 9-11am DCL 3211. Last Class: Genetic Engineering 1. Restriction nucleases 2....
![Page 1: Final Exam: May 4th, 9-11am DCL 3211. Last Class: Genetic Engineering 1. Restriction nucleases 2. DNA labeling 3. Accurate Nucleic acid hybridization,](https://reader034.fdocuments.us/reader034/viewer/2022051401/56649f265503460f94c3d9fe/html5/thumbnails/1.jpg)
Final Exam: May 4th, 9-11am
DCL 3211
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Last Class: Genetic Engineering
1. Restriction nucleases 2. DNA labeling
3. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray
4. Molecular cloning, DNA replication by vector 5. Gene sequencing
6. polymerase chain reaction 7. Monitoring Gene expression
8. the application of genetic engineering: Detect proteins and protein-protein interactions, library
screening, gene mutation
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• Visualizing Cells
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Resolving Power
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Light Microscope
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Interference between light waves
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Resolution Calculation
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Two ways to get contrast
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Four Types of light microscopyBright field, phase contrast, differential
interference contrast, Dark-field microscopy
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Fluorescence Microscope
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Fluorescent Dyes
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Fluorescent imageBlue: DNA; Green: microtubules; Red: centrimere
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Immunofluorescence
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Immunostaining
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Confocal Fluorescence Microscopy
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The difference between conventional and confocal microscopes
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3D reconstruction from confocal images
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Transmission Electron microscopy (TEM, resolution 0.002 nm)
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A root-tip cell under electromicroscopy
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The scanning electron microscope (SEM)
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Stereocillia from a hair cell
SEM
TEMDIC
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Summary of Visualizing Cells
1. Transmitted lights2. Fluorescence
3. Electron microscopy
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Fluorescence Proteins and Live Cell Imaging
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A Cell and A City
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Track Molecular Motions
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Jellyfish and GFP
Osamu Shimomuradiscovered GFP in 1962
Shimomura O, et al, 1962. J. Cell. Comp. Physiol.
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Dr. Douglas Prasher
Prasher DC, et al. 1992. Gene
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Target Molecule GFP
A B
Transcription Translation
488 nm510 nm
Recombinant Gene
Recombinant Protein
GFPTarget Molecule
GFP and its labeling strategy
Wang et al. Annual Review in Biomedical Engineering, 2008
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Martin Chalfie
Chalfie M, et al. 1994. ScienceInouye S, Tsuji FI. 1994. FEBS Lett.
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Passive Applications of GFP
GFP-microtubules
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Sergey A. Lukyanov The Discovery of DsRed (discosoma, coral reef from
Indo-pacific)
Matz MV, et al. 1999. Nature Biotech.
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Roger Y. Tsien
Tsien RY. 1998, Ann Rev Biochem.Tsien RY. 2005, FEBS LettersGiepmans, BN. et al. 2006. Science
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Multiple color visualization
2
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Photoactivatable Fluorescence Proteins
Lukyanov, KA. et al. 2005. Nature Rev Mol Cell Biology
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A BUV
PA
-FP
PA
-FP
UV
PS
-FP
PS
-FP
C UV
Dro
np
a
Blue D
ron
pa
Photoactivatable Fluorescence Proteins
Wang et al. Annual Review in Biomedical Engineering, 2008
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Photoactivatable ProteinsDronpa
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A
FP
144 145
cpF
P
CcpFP
145-238 1-144
Breakage Site
FP
N C
N cpF
P
cpF
P
cpF
P
cpF
P
B
NC
NC
C
Inserted Domain
Stimulator
Stimulator
Domains for interaction
Circularly Permutated Proteins
Wang et al. Annual Review in Biomedical Engineering, 2008
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Calcium Oscillation in Heart
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Technologies utilizing FPs
1. Fluorescence Lifetime Microscopy (FLIM)2. Chromophore Assisted Laser Inactivation (CALI)
3. Fluorescence Resonance Energy Transfer (FRET)4. Applications of FRET Biosensors
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B
Flu
ore
scen
ceIn
ten
sity
Time
Excitation Emission
Time DomainA
Excitation Emission
Frequency Domain
Flu
ore
scen
ceIn
ten
sity
Time
Wang et al. Annual Review in Biomedical Engineering, 2008
Fluorescence Lifetime Microscopy (FLIM)
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FP
Target Molecule
ROS FP
Chromophore Assisted Laser Inactivation (CALI)
Wang et al. Annual Review in Biomedical Engineering, 2008
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Spy on their Actions!
FRET
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The Principle of Fluorescence Resonance Energy Transfer (FRET)
When the fluorophores are far apart: No FRET
Excitation Emission
When fluorophores are close: FRET occurs
Excitation Emission
FRET
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The General Design of FRET-based Fluorescent Probes
EC
FP
EYFP
EYFP
ECFPECFP
A
433 nm
476 nm433 nm
527 nm
FRET
476 nm 433 nm527 nm
EYFP
433 nm
C
B476 nm433 nm
433 nm
ECFP EYFP
EYFP
ECFP
527 nmE
CF
P
EY
FP
Wang et al. Annual Review in Biomedical Engineering, 2008
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FRET-Based BiosensorsCalcium Ras and Rap1
Miyawaki, et al 1997, Nature
Mochizuki, et al 2001, Nature
Ting, et al 2001, PNAS
Tyrosine Kinase Abl
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Why Src?
Src plays a significant role in:
• Cell polarity
• Adhesion
• Focal adhesion dynamics
• Lamellipodia formation
• Migration
• Mechanotransduction
• Cancer development
The first protein tyrosine kinase discovered.
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Design Strategy
Weak FRET
Phosphatase
Strong FRET
433 nm
527 nm
433 nm
490 nm
ECFP(1-227) SH2(from c-Src) Substrate EYFPLinker
Src Activation
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Em
iss i
on
Inte
ns i
ty
Arb
itra
ry U
nit
s
Wavelength (nm)
-Src
+Src
Emission spectra of the Src reporter
The Src kinase induces a FRET response of the Src reporter
CFP YFP
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EGF induced FRET responses in HeLa Cells
Rat
io (
CF
P/Y
FP
)
0.4
0.3
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The Src reporter with CFP and YFP monomers
ECFP(1-227) SH2(from c-Src) Substrate EYFPLinker
A206K A206K
A206K
A206K
0.5
0.35
Zacharias, D. A. et al, Science, 2002
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Construction of membrane-tethered Src reporter
MGCIKSKRKDNLNDDE mCFP SH2 substrate mYFP
mCFP mYFPGC
Plasma Membrane
0.5
0.3
Zacharias, D. A. et al, Science, 2002
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Application of Mechanical Stimulation by Using Laser Tweezers
F1
F2
F
Physical Principle of Laser Tweezers
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Bead
Polystyrene beads were coated with fibronectin and positioned on cells
Cell Body
(with Src reporters)
F
Optic Lens
Light
Integrins
Fibronectin
Actin
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Polystyrene beads were coated with fibronectin and positioned on cells
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Pulling Polylysine-coated beads did not have significant effects on FRET
0.55
0.35
FRET
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Pulling Fibronectin-coated Beads induced a directed and long-range Src activation
Overlay
Force
0.44
0.22
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Pulling Fibronectin-coated Beads induced a directed propagation of Src activation
0.52
0.25
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The directed and long-range activation of Src is dependent on cytoskeleton-integrity
0.45
0.25
Cytochalasin D Treated
0.44
0.25
Nocodazole Treated
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Summary
FRET-based biosensors can allow the detection of various biochemical signals with high tempo-spatial resolution in live cells, including the signal transduction in response to mechanical stimulation.