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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

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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

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


The vision:Bacterial targeting

via membrane display


The vision:Inter-cellular activation

via Lux quorum-sensing


The vision:Intra-cellular activation

via Fec signal transduction


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


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

  • Direct Selection

    • Direct magnetic beads

  • Indirect selection

    • MACS

    • FACS


Direct Selection using Magnetic Beads

After magnetic

selection


Direct Selection using Magnetic Beads


Cell-Cell Signaling:luxI/luxR Quorum Sensing

Reporter

Target

(bead)

Receiver

+

Sender

R

OHHL


Cell-Cell Signaling:Constructs

Sender

Receiver

Single Cell Construct – “JT”

Two Cell Construct

Receiver

Sender


Sharp increase in fluorescence indicates quorum activity

Fluorescence per cell

Amount of sender cells added


Testing for self-induction: Fluorescence over OD at various times


Direct Magnetic Beads: Good Enrichment


The plate-drop experiment

BBa_S03623 – BBa_I13507

BBa_T9002

T9002

S23I07

OHHL Receiver -> GFP

Red OHHL sender


Plate Drop Experiment with Enriched Sender


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

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

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

  • 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

  • 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

To be added


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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