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Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction nucleases 5. Molecular cloning, DNA replication by vector 6. polymerase chain reaction 7. Monitoring Gene expression 8. the application of genetic engineering: Detect proteins and protein-protein interactions,

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Page 1: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Last Class: Genetic Engineering

1. DNA labeling2. Accurate Nucleic acid hybridization,

Northern/Southern Blot, Microarray 3. Gene sequencing

4. Restriction nucleases 5. Molecular cloning, DNA replication by vector

6. polymerase chain reaction 7. Monitoring Gene expression

8. the application of genetic engineering: Detect proteins and protein-protein interactions, library

screening, gene mutation

Page 2: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

• Visualizing Cells

Page 3: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Resolving Power

Page 4: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Light Microscope

Page 5: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Interference between light waves

Page 6: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Two ways to get contrast

Page 7: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Resolution Calculation

Page 8: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Four Types of light microscopyBright field, phase contrast, differential

interference contrast, Dark-field microscopy

Page 9: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Immunostaining

Page 10: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Fluorescence Microscope

Page 11: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Fluorescent Dyes

Page 12: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Fluorescent imageBlue: DNA; Green: microtubules; Red: centrimere

Page 13: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Immunofluorescence

Page 14: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Confocal Fluorescence Microscopy

Page 15: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

The difference between conventional and confocal microscopes

Page 16: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

3D reconstruction from confocal images

Page 17: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Transmission Electron microscopy (TEM, resolution 0.002 nm)

Page 18: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

A root-tip cell under electromicroscopy

Page 19: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

The scanning electron microscope (SEM)

Page 20: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Stereocillia from a hair cell

SEM

TEMDIC

Page 21: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Summary of Visualizing Cells

1. Transmitted lights2. Fluorescence

3. Electron microscopy

Page 22: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Fluorescent Proteins and Live Cell Imaging

Page 23: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

A Cell and A City

Page 24: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Track Molecular Motions

Page 25: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Jellyfish and GFP

Osamu Shimomuradiscovered GFP in 1962

Shimomura O, et al, 1962. J. Cell. Comp. Physiol.

Page 26: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Dr. Douglas Prasher

Prasher DC, et al. 1992. Gene

Page 27: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

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

Page 28: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Martin Chalfie

Chalfie M, et al. 1994. ScienceInouye S, Tsuji FI. 1994. FEBS Lett.

Page 29: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Passive Applications of GFP

GFP-microtubules

Page 30: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Sergey A. Lukyanov The Discovery of DsRed (discosoma, coral reef from

Indo-pacific)

Matz MV, et al. 1999. Nature Biotech.

Page 31: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Roger Y. Tsien

Tsien RY. 1998, Ann Rev Biochem.Tsien RY. 2005, FEBS LettersGiepmans, BN. et al. 2006. Science

Page 32: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Multiple color visualization

2

Page 33: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Photoactivatable Fluorescence Proteins

Lukyanov, KA. et al. 2005. Nature Rev Mol Cell Biology

Page 34: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

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

Page 35: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Photo-activatable ProteinsDronpa

Page 36: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

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

Page 37: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Calcium Oscillation in Heart

Page 38: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction
Page 39: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

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

Page 40: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

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)

Page 41: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

FP

Target Molecule

ROS FP

Chromophore Assisted Laser Inactivation (CALI)

Wang et al. Annual Review in Biomedical Engineering, 2008

Page 42: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Spy on their Actions!

FRET

Page 43: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

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

Page 44: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

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

Page 45: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

FRET-Based BiosensorsCalcium Ras and Rap1

Miyawaki, et al 1997, Nature

Mochizuki, et al 2001, Nature

Ting, et al 2001, PNAS

Tyrosine Kinase Abl

Page 46: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

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.

Page 47: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

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

Page 48: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

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

Page 49: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

EGF induced FRET responses in HeLa Cells

Rat

io (

CF

P/Y

FP

)

0.4

0.3

Page 50: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

The Src reporter with CFP and YFP monomers

ECFP(1-227) SH2(from c-Src) Substrate EYFPLinker

A206K A206K

A206K

A206K

Zacharias, D. A. et al, Science, 2002

0.5

0.35

Page 51: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

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

Page 52: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Application of Mechanical Stimulation by Using Laser Tweezers

F1

F2

F

Physical Principle of Laser Tweezers

Page 53: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Pulling Polylysine-coated beads did not have significant effects on FRET

0.55

0.35

FRET

Page 54: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Bead

Polystyrene beads were coated with fibronectin and positioned on cells

Cell Body

(with Src reporters)

F

Optic Lens

Light

Integrins

Fibronectin

Actin

Page 55: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Polystyrene beads were coated with fibronectin and positioned on cells

Page 56: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Pulling Fibronectin-coated Beads induced a directed propagation of Src activation

0.52

0.25

Page 57: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

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

Page 58: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

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.

Page 59: Last Class: Genetic Engineering 1. DNA labeling 2. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 3. Gene sequencing 4. Restriction

Pulling Fibronectin-coated Beads induced a directed and long-range Src activation

Overlay

Force

0.44

0.22


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