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

Harvard iGEM 2007Introduction

Bind Proteins

Bind Other Cells

Bind Tissue

Bind SurfaceBind DNA

Bind Viruses

Bind Toxins

Potential Targets and Applications

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

Selecting/enriching for surface engineered bacteria

• Direct Selection– Direct magnetic beads

• Indirect selection– MACS– FACS

Figure here to illustrate Direct selection and indirect selection

Harvard iGEM 2007Bacterial Targeting

Cell Sorting with his and strep2

Indirect Bead Assays

Direct Bead Assays

Harvard iGEM 2007Bacterial Targeting

<NEW TITLE>Test: Cell Sorting with

AIDA1 + sender constructs (with his

and strep2)

his

strep2

Before Separation

After Separation

Harvard iGEM 2007Bacterial Targeting

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

Harvard iGEM 2007Quorum Sensing

luxI/luxR Quorum Sensing

Receiver

Sender+

ReporterR

OHHL

Harvard iGEM 2007Quorum Sensing

• Receivers (luxR + Reporter)– GFP Receivers (Bba_T9002)– mRFP Receivers (Bba_F2620 + 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)

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

Harvard iGEM 2007Quorum Sensing

Switch-like Quorum Response

Sender

Receiver

Harvard iGEM 2007Quorum Sensing

Selection with Direct Magnetic Beads

Control: no selection

Experimental: Selection with beads

Harvard iGEM 2007Quorum Sensing

60-fold Enrichment through Direct Magnetic Beads

Control: no beads Selection with streptactin beads

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 MethodsStructural 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

• Targeting: AIDA

• Intercellular signaling: QS

• Intracellular signaling: Fec

Harvard iGEM 2007Conclusion

Future Directions

• Targeting: AIDA

• Intercellular signaling: QS

• Intracellular signaling: Fec

Harvard iGEM 2007Conclusion

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

Harvard iGEM 2007Bacterial Targeting

N terminus modification of AIDA1

MACS Results

white

white

red red 0

102030405060708090

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aida1+strep2 aida1+his

Cell Types

Co

lon

y co

un

t

Harvard iGEM 2007Bacterial Targeting

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

Harvard iGEM 2007Bacterial Targeting

CSR by gene design

Harvard iGEM 2007Bacterial Targeting

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