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 motivationTo develop a system for directing bacteria

to a target of interest and effecting downstream activity


Bind Proteins

Bind Other Cells

Bind Tissue

Bind SurfaceBind DNA

Bind Viruses

Bind Toxins

Potential Targets and Applications


Quorum-sensing Fec signal transduction

Bacterial targeting


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


Selecting/enriching for surface engineered bacteria

• Direct Selection– Direct magnetic beads

• Indirect selection– MACS– FACS

Figure here to illustrate Direct selection and indirect selection


Cell Sorting with his and strep2

Indirect Bead Assays

Direct Bead Assays


<NEW TITLE>Test: Cell Sorting with

AIDA1 + sender constructs (with his

and strep2)

his

strep2

Before Separation

After Separation


Results: (MORE DESCRIPTIVE TITLE HERE)

Successful selection of E.coli expressing His or Strep2 on surface

Initial cultures Enriched cultures through selection

Harvard iGEM 2007Quorum Sensing

Bacterial targeting

Fec signal transductionQuorum-sensing


luxI/luxR Quorum Sensing

Receiver

Sender+

ReporterR

OHHL


• Receivers (luxR + Reporter)– GFP Receivers

• tetR controlled (Bba_T9002)• Quorum controlled (Bba_R0062 + Bba_C0261 + Bba_E0240)

– mRFP Receivers • tetR controlled (Bba_F2620 + Bba_I13507)• Quorum controlled (Bba_R0062 + Bba_C0261 + Bba_I13507)

– mCherry Receivers (Bba_F2620 + Bba_J06702)• Senders (bicistronic luxI + Reporter)

– mRFP Sender • tetR controlled (Bba_S03623 + Bba_I13507)• lacI controlled (Bba_S03608 + Bba_I13507)• Quorum controlled (Bba_R0062 + Bba_A340620 + Bba_I13507)

– GFP Sender• tetR controlled (Bba_S03623 + Bba_E0240)• lacI controlled (Bba_S03608 + Bba_E0240)• Quorum controlled (Bba_R0062 + Bba_A340620 + Bba_E0240)

– mCherry Sender• tetR controlled (Bba_S03623 + Bba_J06702)

• Single Cell– Constitutive (Bba_J23039 + Bba_T9002)– Quorum Controlled (Bba_R0062 + Bba_A340620 + Bba_C0261 + Bba_E0240)

• Construction Intermediates

Cell-Cell Signaling ConstructsReceiver

Sender


Switch-like Quorum Response

Sender

Receiver


Selection with Direct Magnetic Beads

Control: no selection

Experimental: Selection with beads


60-fold Enrichment through Direct Magnetic Beads

Control: no beads Selection with streptactin beads


The plate-drop experiment

Receiver Sender

Harvard iGEM 2007Two Component System

Bacterial targeting



Motivation: Fec System

• Goal: Direct cell signaling

• Method: Re-engineer an existing signaling pathway

• Fec system:– well-characterized– substrate specific


Overview of Fec System

Braun et al. “Gene Regulation by Transmembrane Signaling.” Biometals 2006 Apr;19(2):103-13.


Fec: Motivation and Methods• Re-engineer FecA: Mutate

loop 7 and/or loop 8• Structural Evidence:

– L7 moves up to 11Å, helix unwinds

– L8 moves up to 15Å• Assume signaling will

occur with binding.

Ferguson AD et al. “Structural Basis of Gating by the Outer Membrane Transporter FecA. Science 2002 Mar 1: 295(5560) 1715-9

Loops 7 & 8


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.


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 2007Conclusion

CONCLUSION

• Targeting: AIDA

• Intercellular signaling: QS

• Intracellular signaling: Fec


Future Directions

• Targeting: AIDA

• Intercellular signaling: QS

• Intracellular signaling: Fec


ACKNOWLEDGEMENTS

AdvisorsGeorge Church

Debra Auguste

Jagesh V. Shah

William Shih

Pamela Silver

Alain Viel

Tamara Brenner

Teaching FellowsNicholas Guido

Bill Senapedis

Mike Strong

Harris Wang

FundingHHMI

Harvard Provost

Harvard Life Sciences Division

Harvard School of Engineering and Applied Sciences


N terminus modification of AIDA1

MACS Results

white

white

red red 0

102030405060708090

100

aida1+strep2 aida1+his

Cell Types

Co

lon

y co

un

t


CSR by gene Design

• Fusion of tags & randomers to extracellular portion of OmpA loop 1 (loop insertion)– PCR product insertion (950 bps)– Insertion of ds oligos


CSR by gene design


Bacterial Targeting: Cell Surface Reengineering (CSR)

• CSR by PCR product digestion & ligation:– Fusion of peptides to the C terminus of OmpA– Fusion of peptides to the N terminus of AIDA1

• <OmpA AIDA1 structures>• Fusion of tags & randomers to extracellular portion

of OmpA loop 1 (loop insertion)– PCR product insertion (950 bps)– Insertion of ds oligos

Cling- E. coli : Bacteria on target

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