Download Cling-E. coli : Bacteria on target

Cling-E. coli :
Bacteria on target
Harvard iGEM 2007
Ellenor Brown
Stephanie Lo
Alex Pickett
Sammy Sambu
Kevin Shee
Perry Tsai
Shaunak Vankudre
George Xu
The motivation
To develop a system for directing bacteria
to a target of interest and effecting
downstream activity
Introduction
Harvard iGEM 2007
Bacterial targeting
Quorum-sensing
Introduction
Harvard iGEM 2007
Fec signal transduction
Bacterial targeting
Quorum-sensing
Introduction
Harvard iGEM 2007
Fec signal transduction
Bacterial targeting
Quorum-sensing
Introduction
Harvard iGEM 2007
Fec signal transduction
Bacterial targeting
Quorum-sensing
Introduction
Harvard iGEM 2007
Fec signal transduction
Surface Engineered Bacteria
Engineered to Bind and Signal
OmpA – C terminal insertion
Fusion Protein
OmpA-Loop1 insertion
AIDA-1 – N terminal insertion
FecA – loop insertion
Bacterial Targeting
Harvard iGEM 2007
Membrane
Protein
Surface Engineered Bacteria
Engineered
to
Bind
and
Signal
Positive Signal
Background
AIDA-1 his
or
AIDA-1 strep2
Sender LuxI RFP
Kan
Amp and Kan
Amp
Co-transform
signal
Bacterial Targeting
Harvard iGEM 2007
Selecting/enriching for surface
engineered bacteria
• Direct Selection
– Direct magnetic beads
• Indirect selection
– MACS
– FACS
Bacterial Targeting
Harvard iGEM 2007
Direct Selection using Magnetic Beads
After magnetic
selection
Bacterial Targeting
Harvard iGEM 2007
Direct Selection using Magnetic Beads
Bacterial Targeting
Harvard iGEM 2007
Bacterial targeting
Quorum-sensing
Quorum Sensing
Harvard iGEM 2007
Fec signal transduction
Cell-Cell Signaling:
luxI/luxR Quorum Sensing
Reporter
R
Target
(bead)
Receive
r +
Sender
OHHL
Quorum Sensing
Harvard iGEM 2007
Cell-Cell Signaling:
Constructs
Sender
Single Cell Construct – “JT”
Receiver
Two Cell Construct
Receiver
Quorum Sensing
Harvard iGEM 2007
Sender
Sharp increase in fluorescence
indicates quorum activity
GFP Fluorescence per OD
400
350
300
250
200
150
100
50
0
0
20
40
60
80
100
120
140
Amount of Sender Cells Added
Quorum Sensing
Harvard iGEM 2007
160
180
200
Direct Magnetic Beads:
60-fold Enrichment
Quorum Sensing
Harvard iGEM 2007
The plate-drop experiment
BBa_T9002
BBa_S03623 – BBa_I13507
T9002
S23I07
OHHL Receiver -> GFP
Red OHHL sender
Quorum Sensing
Harvard iGEM 2007
Plate Drop Experiment with Enriched
Sender
Quorum Sensing
Harvard iGEM 2007
Bacterial targeting
Quorum-sensing
Two Component System
Harvard iGEM 2007
Fec signal transduction
Direct Signaling from the Outer
Membrane: the Fec System
•
Advantages of Direct Signaling
from the Outer Membrane:
Substrate Specificity
•
The FecIRA system is the only
well-characterized signaling
scaffold in Gram-negative bacteria
•
FecA is an iron transporter and
signal transducer on the outer
membrane of E. Coli K-12
•
When ferric citrate binds, FecA
activates periplasmic FecR, which
then activates the sigma factor
FecI, resulting in gene expression
•
The system is repressed by the
Fur repressor in iron-rich
conditions
Two Component System
Harvard iGEM 2007
Braun et al. “Gene Regulation by Transmembrane Signaling
Biometals 2006 Apr;19(2):103-13.
Fec: Motivation and Methods
•
Structural information suggests
possibility of maintaining signaling with
changed binding.
– L7 moves up to 11Å, helix unwinds
– L8 moves up to 15Å
•
Select binding targets by inserting
random library, controls known to bind
nickel and streptavidin into loops 7 and 8.
– Even if signaling cannot be
maintained, binding of controls
proves that FecA can be used as
scaffold for surface expression of
peptides
•
Computational approach in collaboration
with the lab of Costas Maranas, Penn
State Dept ofSystem
Chemical Engineering.
Two Component
Harvard iGEM 2007
Ferguson AD et al. “Structural Basis of Gating by the Outer Membrane Transport
Science 2002 Mar 1: 295(5560) 1715-9
Results
FecA Induction in Presence of Sodium Citrate in LB
•
•
Wild Type Induction of FecA with
Sodium Citrate and a GFP
Reporter shows approximately
2000 RFU increase
MACS Results
Results from Nickel and His
Fluorescence Assays
2000
1500
RFUs
•
1000
500
0
0:00:00
2:24:00
4:48:00
7:12:00
Time(mins)
Two Component System
Harvard iGEM 2007
9:36:00
12:00:00
14:24:00
Biobricking the Fec System
Construct Features:
•Swappable FecA - FecA is
flanked by Nhe1 and AflII sites
to allow the easy mutagenesis
and replacement of FecA.
•Variable Promoters - each
component will be on a
separate constitutive promoter.
•The optimization of GFP
expression using promoters of
different strengths is planned.
Two Component System
Harvard iGEM 2007
Biobricking the Fec System
• Mutagenesis of Fec promoter
to weaken gene expression,
providing a range of sensitivity.
•Mutagenesis of the Fec
promoter to remove FUR
repressor binding site, allowing
easier assays.
Two Component System
Harvard iGEM 2007
CONCLUSION
• To be added
Conclusion
Harvard iGEM 2007
ACKNOWLEDGEMENTS
Advisors
Teaching Fellows
George Church
Debra Auguste
Jagesh V. Shah
William Shih
Pamela Silver
Alain Viel
Tamara Brenner
Nicholas Guido
Bill Senapedis
Mike Strong
Harris Wang
Conclusion
Harvard iGEM 2007