Final Exam: May 4th, 9-11am

DCL 3211

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

• Visualizing Cells

Resolving Power

Light Microscope

Interference between light waves

Resolution Calculation

Two ways to get contrast

Four Types of light microscopyBright field, phase contrast, differential

interference contrast, Dark-field microscopy

Fluorescence Microscope

Fluorescent Dyes

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

Immunofluorescence

Immunostaining

Confocal Fluorescence Microscopy

The difference between conventional and confocal microscopes

3D reconstruction from confocal images

Transmission Electron microscopy (TEM, resolution 0.002 nm)

A root-tip cell under electromicroscopy

The scanning electron microscope (SEM)

Stereocillia from a hair cell

SEM

TEMDIC

Summary of Visualizing Cells

1. Transmitted lights2. Fluorescence

3. Electron microscopy

Fluorescence Proteins and Live Cell Imaging

A Cell and A City

Track Molecular Motions

Jellyfish and GFP

Osamu Shimomuradiscovered GFP in 1962

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

Dr. Douglas Prasher

Prasher DC, et al. 1992. Gene

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

Martin Chalfie

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

Passive Applications of GFP

GFP-microtubules

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

Indo-pacific)

Matz MV, et al. 1999. Nature Biotech.

Roger Y. Tsien

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

Multiple color visualization

2

Photoactivatable Fluorescence Proteins

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

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

Photoactivatable ProteinsDronpa

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

Calcium Oscillation in Heart

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

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)

FP

Target Molecule

ROS FP

Chromophore Assisted Laser Inactivation (CALI)

Wang et al. Annual Review in Biomedical Engineering, 2008

Spy on their Actions!

FRET

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

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

FRET-Based BiosensorsCalcium Ras and Rap1

Miyawaki, et al 1997, Nature

Mochizuki, et al 2001, Nature

Ting, et al 2001, PNAS

Tyrosine Kinase Abl

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.

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

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

EGF induced FRET responses in HeLa Cells

Rat

io (

CF

P/Y

FP

)

0.4

0.3

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

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

Application of Mechanical Stimulation by Using Laser Tweezers

F1

F2

F

Physical Principle of Laser Tweezers

Bead

Polystyrene beads were coated with fibronectin and positioned on cells

Cell Body

(with Src reporters)

F

Optic Lens

Light

Integrins

Fibronectin

Actin

Polystyrene beads were coated with fibronectin and positioned on cells

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

0.55

0.35

FRET

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

Overlay

Force

0.44

0.22

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

0.52

0.25

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

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.