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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.

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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
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

slide3
The vision:Bacterial targeting

via membrane display

slide5
The vision:Intra-cellular activation

via Fec signal transduction

slide6
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

slide7
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

selecting enriching for surface engineered bacteria
Selecting/enriching for surface engineered bacteria
  • Direct Selection
    • Direct magnetic beads
  • Indirect selection
    • MACS
    • FACS
cell cell signaling luxi luxr quorum sensing
Cell-Cell Signaling:luxI/luxR Quorum Sensing

Reporter

Target

(bead)

Receiver

+

Sender

R

OHHL

cell cell signaling constructs
Cell-Cell Signaling:Constructs

Sender

Receiver

Single Cell Construct – “JT”

Two Cell Construct

Receiver

Sender

sharp increase in fluorescence indicates quorum activity
Sharp increase in fluorescence indicates quorum activity

Fluorescence per cell

Amount of sender cells added

slide16
The plate-drop experiment

BBa_S03623 – BBa_I13507

BBa_T9002

T9002

S23I07

OHHL Receiver -> GFP

Red OHHL sender

direct signaling from the outer membrane the fec 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.

fec motivation and methods
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

results
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

biobricking the fec system

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.
biobricking the fec system1

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.
conclusion
CONCLUSION

To be added

acknowledgements
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

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