Cling- E. coli : Bacteria on target

Cling-E. coli :Bacteria on target

Harvard iGEM 2007Ellenor BrownStephanie LoAlex PickettSammy Sambu

Kevin SheePerry TsaiShaunak VankudreGeorge Xu

Harvard iGEM 2007Introduction

The motivation

To develop a system for directing bacteria to a target of interest and effecting

downstream activity

Harvard iGEM 2007Introduction

Quorum-sensing Fec signal transduction

Bacterial targeting

Quorum-sensing 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

Bacterial targeting

Fec signal transductionQuorum-sensing

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

Harvard iGEM 2007Quorum Sensing

Direct Magnetic Beads: 60-fold 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

Bacterial targeting

Quorum-sensing Fec signal transduction

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