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!