fret microscopy presentation
TRANSCRIPT
Z-RING DYNAMICS INVESTIGATED BY
FRET MICROSCOPY
Vallery Salomon
PRESENTATION OUTLINE
Introduction
Materials and Methods
Results
Image Analysis
Discussion
INTRODUCTION
INTRODUCTION
Cell Division
WHY FRET MICROSCOPY?
INTRODUCTION
Forster Resonance Energy Transfer (FRET)
INTRODUCTION
MATERIALS AND METHODS
MATERIALS AND METHODS
The strain of bacteria we used for this experiment is
E. Coli with FtsZ-CFP and FtsZ-YFP(PZW166)
MATERIALS AND METHODS
Growth Condition
- Grow PZW166 at 37C in 20ml LB for 12hrs.
MATERIALS AND METHODS
Plasma Treatment of
PDMS
Coat microchannel
with Bovine Serum
Albumin (BSA)
Inject cells into
microchannel
MATERIALS AND METHODS
Inlet
Outlet
MATERIALS AND METHODS
Load cells by centrifuging
Grow cells in fresh medium
MATERIALS AND METHODS
Set up Microscope
Microscopy
RESULTS
RESULTS
Video of Donor, Acceptor, and FRET signals.
RESULTS
FRET Acceptor Donor
IMAGE ANALYSIS
FRET Acceptor Donor
IMAGE ANALYSIS
Mean + 3 std
• Cut off background noise
IMAGE ANALYSIS
• Rotate and Crop Z-rings
IMAGE ANALYSIS
RESULTS
The donor signal is supposed to be decreasing but
it is not
RESULTS
The acceptor signal is supposed to be straight but it
is not
RESULTS
The FRET signal is supposed to be increasing but it
is not
DISCUSSION
Improvements of experimental method:
-Concentration of inducer
-Longer wait period between injection
and microscopy
DISCUSSION
PFRET=uFRET-ASBT-DSBT
Improvements of image analysis:
DISCUSSION
We have another strain of bacteria we use that is
only FtsZ-CFP (PZW36)
We have another strain of bacteria we use that is
only FtsZ-YFP (PZW79)
SOURCES
Quantitation of Protein-Protein Interaction, Methods
In Cell Biology, A. Periasamy (2008)
Robust Growth of Escherichia Coli, Current Biology,
P. Wang (2010)
Direct interactions of early and late assembling
division proteins in Escherichia coli cells resolved
by FRET, Molecular Microbiology, S. Alexeeva
(2010)
THANK YOU FOR AN AMAZING SUMMER!