Cling-E. coli : Bacteria on target

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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
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The motivation
To develop a system for directing bacteria to a
target of interest and effecting downstream
activity

• 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

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Bacterial targeting
Quorum sensing
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The visionBacterial targeting via membrane
display
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The visionInter-cellular activation via Lux
quorum-sensing
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The visionIntra-cellular activation via Fec
signal transduction
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Surface Engineered Bacteria Engineered to Bind
and Signal
Fusion Protein
OmpA C terminal insertion OmpA-Loop1
insertion AIDA-1 N terminal insertion FecA
loop insertion
Membrane Protein
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Surface Engineered Bacteria Engineered to Bind
and Signal
Positive Signal
Background
AIDA-1 his or AIDA-1 strep2
Sender LuxI RFP
Amp and Kan
Kan
Amp
Co-transform
signal
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Selecting/enriching for surface engineered
bacteria

• Direct Selection
• Direct magnetic beads
• Indirect selection
• MACS
• FACS

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Direct Selection using Magnetic Beads
After magnetic selection
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Direct Selection using Magnetic Beads
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Cell-Cell SignalingluxI/luxR Quorum Sensing

Receiver

Sender
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Cell-Cell SignalingConstructs
Receiver
Sender
Single Cell Construct JT
Two Cell Construct
Sender
Receiver
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Sharp increase in fluorescence indicates quorum
activity
Fluorescence per cell
Amount of sender cells added
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Testing for self-induction Fluorescence over OD
at various times
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Direct Magnetic Beads Good Enrichment
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The plate-drop experiment
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Plate Drop Experiment with Enriched Sender
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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 Apr19(2)103-13.
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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
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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

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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.

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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.

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CONCLUSION

• To be added

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ACKNOWLEDGEMENTS
Advisors George Church Debra Auguste Jagesh V.
Shah William Shih Pamela Silver Alain Viel Tamara
Brenner
Teaching Fellows Nicholas Guido Bill
Senapedis Mike Strong Harris Wang