This document outlines an experiment that used FRET microscopy to investigate the dynamics of the Z-ring in E. coli cells. The experiment used E. coli strains containing both FtsZ-CFP and FtsZ-YFP to observe FRET signals during cell division. Results showed donor, acceptor, and FRET signals over time, but the patterns did not match expectations, indicating issues with the experimental or analysis methods. Improvements discussed including changing inducer concentration and wait time before microscopy, as well as improving the image analysis methodology.

1. Z-RING DYNAMICS INVESTIGATED BY
FRET MICROSCOPY
Vallery Salomon

2. PRESENTATION OUTLINE
 Introduction
 Materials and Methods
 Results
 Image Analysis
 Discussion

3. INTRODUCTION

4. INTRODUCTION
 Cell Division

5. WHY FRET MICROSCOPY?

6. INTRODUCTION
 Forster Resonance Energy Transfer (FRET)

7. INTRODUCTION

8. MATERIALS AND METHODS

9. MATERIALS AND METHODS
 The strain of bacteria we used for this experiment is
E. Coli with FtsZ-CFP and FtsZ-YFP(PZW166)

10. MATERIALS AND METHODS
 Growth Condition
- Grow PZW166 at 37C in 20ml LB for 12hrs.

11. MATERIALS AND METHODS
 Plasma Treatment of
PDMS
 Coat microchannel
with Bovine Serum
Albumin (BSA)
 Inject cells into
microchannel

12. MATERIALS AND METHODS
Inlet
Outlet

13. MATERIALS AND METHODS
 Load cells by centrifuging
 Grow cells in fresh medium

14. MATERIALS AND METHODS
 Set up Microscope
 Microscopy

15. RESULTS

16. RESULTS
 Video of Donor, Acceptor, and FRET signals.

17. RESULTS
FRET Acceptor Donor

18. IMAGE ANALYSIS
FRET Acceptor Donor

19. IMAGE ANALYSIS
Mean + 3 std
• Cut off background noise

20. IMAGE ANALYSIS
• Rotate and Crop Z-rings

21. IMAGE ANALYSIS

22. RESULTS
 The donor signal is supposed to be decreasing but
it is not

23. RESULTS
 The acceptor signal is supposed to be straight but it
is not

24. RESULTS
 The FRET signal is supposed to be increasing but it
is not

25. DISCUSSION
 Improvements of experimental method:
-Concentration of inducer
-Longer wait period between injection
and microscopy

26. DISCUSSION
PFRET=uFRET-ASBT-DSBT
Improvements of image analysis:

27. 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)

28. 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)

29. THANK YOU FOR AN AMAZING SUMMER!