harvard igem 2007 introduction cling-e. coli : bacteria on target harvard igem 2007 ellenor brown...

Harvard iGEM 2007Introduction

Cling-E. coli :Bacteria on target

Harvard iGEM 2007Ellenor BrownStephanie LoAlex PickettSammy Sambu

Kevin SheePerry TsaiShaunak VankudreGeorge Xu


The motivationTo develop a system for targeting bacteria

to a specific substrate and effecting a cellular response


Bind Proteins

Bind Other Cells

Bind Tissue

Bind Surface

Bind DNA/RNA

Bind Viruses

Bind Toxins

Potential Targets and Applications


Quorum-sensing Fec signal transduction

Bacterial targeting



Surface Engineered BacteriaEngineered to Bind and Signal

Fusion Protein

Membrane Protein

OmpA – C terminal insertion

OmpA-Loop1 insertion

AIDA-1 – N terminal insertion


Selecting/enriching for surface engineered bacteria

• Tags– 6xHis + nickel beads– Strep2 + streptavidin

beads

• Assays– Magnetic Activated Cell

Sorting (MACS)– Fluorescence Activated

Cell Sorting (FACS)Fluorescence Bead Assays

Magnetic Bead Assays


His/Strep2 –tagged bacteria are enriched by MACS

(after MACS selection)


Results:

Cell Selection Assays are a Success! AIDA was re-engineered to target nickel and streptavidin with 6xHis and Strep2 tags

respectively, and selecting for surface-engineered bacteria was accomplished through magnetic activated cell sorting.

Harvard iGEM 2007Quorum Sensing

Bacterial targeting

Fec signal transductionQuorum-sensing


luxI/luxR Quorum Sensing

Sender

+

R

OHHL

Receiver


• 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

ReceiverR


Selection with Magnetic Beads

Sender

AIDA-1 – N terminal insertion


60-fold Selection through Magnetic Beads

Control: no beads Selection with streptavidin beads

Green (untagged)

Red (tagged)


Enriched senders activate quorum response

Receiver Sender

OHHL

Harvard iGEM 2007Fec signal transduction

Bacterial targeting



Motivation: Fec System

• Goal: Direct cell signaling

• Method: Re-engineer an existing signal transduction 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.

Ferric citrate


Overview of Fec SystemFerric citrate

Loops 7 & 8


Constructs

• From Braun lab (U. Tuebingen, Germany)– Fec knock-out strain, AA93– FecIRA plasmid– Fec promoter, GFP plasmid

• pColA Duet Vector– Allows regulated expression of Fec genes

under T7 promoter


Wild-Type GFP ExpressionGFP Fluorescence Assays

10mM Sodium Citrate, AA93 Co-transformed

Constitutive GFP

Un-induced AA93

0

200

400

600

800

1000

1200

1400

1600

1800

2000

0:00 2:24 4:48 7:12 9:36 12:00 14:24

time (hh:mm)

RF

Us


Troubleshooting andNext Steps

• Problems:– Growth media– Toxicity: membrane disruption?

• Goals:– Nickel and Streptavidin Binding– Finding new targets with signaling

• Random library• Computational Approach

Harvard iGEM 2007Conclusion

Conclusions• Targeting

– His and Strep2 tags on AIDA, targeting bacteria to nickel and streptavidin was successful

• Quorum sensing– Constructed one-cell system– Characterized two-cell system– Combined with targeting

• Fec signal transduction– Characterized Fec system


Future Directions• Bacterial targeting

– Trying out new targeting peptides (calmodulin)– Optimizing the random library approach in selecting for targeting

peptides

• Quorum sensing– Characterizing one-cell system– Optimizing quorum response after targeting

• Fec signal transduction– Effect signal transduction with targeting

• Application


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

FundingHHMI

Harvard Provost

Harvard Life Sciences Division

Harvard School of Engineering and Applied Sciences

Special thanks to…Volkmar Braun

(University of Tuebingen)

Costas Maranas (Penn State University)

Harvard iGEM 2007Bacterial Targeting

N terminus modification of AIDA1

MACS Results

white

white

red red 0

102030405060708090

100

aida1+strep2 aida1+his

Cell Types

Co

lon

y c

ou

nt


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

harvard igem 2007 introduction cling-e. coli : bacteria on target harvard igem 2007 ellenor brown...

Documents

r0062 bba

a340620 bba

s03623 bba

c0261 bba

f2620 bba

s03608 bba

j23039 bba

t9002quorum controlled