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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. - PowerPoint PPT Presentation
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Cling-E. coli :Bacteria on target
Harvard iGEM 2007Ellenor BrownStephanie LoAlex PickettSammy Sambu
Kevin SheePerry TsaiShaunak VankudreGeorge Xu
Harvard iGEM 2007Introduction
The motivation
• Bacterial targeting is necessary for spatially-specific activity in the body or in nature
• Post-targeting activity and transmembrane signalling are the next step in engineering genetic circuits that interface extracellular and intracellular environments
To develop a system for directing bacteria to a target of interest and effecting
downstream activity
Harvard iGEM 2007Introduction
The vision:Bacterial targeting
via membrane display
Harvard iGEM 2007Introduction
The vision:Inter-cellular activation
via Lux quorum-sensing
Harvard iGEM 2007Introduction
The vision:Intra-cellular activation
via Fec signal transduction
Harvard iGEM 2007Bacterial Targeting
Surface Engineered BacteriaEngineered to Bind and Signal
Fusion Protein
Membrane Protein
OmpA – C terminal insertion
OmpA-Loop1 insertion
AIDA-1 – N terminal insertion
FecA – loop insertion
Harvard iGEM 2007Bacterial Targeting
AIDA-1 hisorAIDA-1 strep2
Surface Engineered BacteriaEngineered to Bind and Signal
Kan
Sender LuxI RFP
Amp Amp and Kan
Positive Signal Background
Co-transform
signal
Harvard iGEM 2007Bacterial Targeting
Selecting/enriching for surface engineered bacteria
• Direct Selection– Direct magnetic beads
• Indirect selection– MACS– FACS
Harvard iGEM 2007Bacterial Targeting
After magneticselection
Direct Selection using Magnetic Beads
Harvard iGEM 2007Bacterial Targeting
Direct Selection using Magnetic Beads
Harvard iGEM 2007Quorum Sensing
Cell-Cell Signaling:luxI/luxR Quorum Sensing
Receiver
Sender
R
OHHL
Target(bead)
Reporter
+
Harvard iGEM 2007Quorum Sensing
Cell-Cell Signaling:Constructs
SenderReceiver
Single Cell Construct – “JT”
Two Cell Construct
ReceiverSender
Harvard iGEM 2007Quorum Sensing
Sharp increase in fluorescence indicates quorum activity
Amount of sender cells added
Fluorescence per cell
Harvard iGEM 2007Quorum Sensing
Testing for self-induction: Fluorescence over OD at various times
0
500
1000
1500
2000
2500
3000
Nondiluted JT 1in200 dilution T02 nondilute
Flu
ore
scen
ce o
ver
OD
0
20
40
60
80
100
120
140
160
180
200
220
240
After Overnight
Harvard iGEM 2007Quorum Sensing
Direct Magnetic Beads: Good Enrichment
Harvard iGEM 2007Quorum Sensing
BBa_T9002
The plate-drop experiment
BBa_S03623 – BBa_I13507
T9002
OHHL Receiver -> GFP
S23I07
Red OHHL sender
Harvard iGEM 2007Quorum Sensing
Plate Drop Experiment with Enriched Sender
Harvard iGEM 2007Two Component System
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
Braun et al. “Gene Regulation by Transmembrane Signaling.” Biometals 2006 Apr;19(2):103-13.
Harvard iGEM 2007Two Component System
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 of Chemical Engineering.
Ferguson AD et al. “Structural Basis of Gating by the Outer Membrane Transporter FecA. Science 2002 Mar 1: 295(5560) 1715-9
Harvard iGEM 2007Two Component System
Results
• 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
FecA Induction in Presence of Sodium Citrate in LB
0
500
1000
1500
2000
0:00:00 2:24:00 4:48:00 7:12:00 9:36:00 12:00:00 14:24:00
Time(mins)
RFU
s
Harvard iGEM 2007Two Component 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.
Biobricking the Fec System
Harvard iGEM 2007Two Component 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.
Biobricking the Fec System
Harvard iGEM 2007Conclusion
CONCLUSION
• To be added
Harvard iGEM 2007Conclusion
ACKNOWLEDGEMENTSAdvisors
George Church
Debra Auguste
Jagesh V. Shah
William Shih
Pamela Silver
Alain Viel
Tamara Brenner
Teaching FellowsNicholas Guido
Bill Senapedis
Mike Strong
Harris Wang