Cling-E. coli :Bacteria on target

Harvard iGEM 2007Ellenor BrownStephanie LoAlex PickettSammy Sambu

Kevin SheePerry TsaiShaunak VankudreGeorge Xu

The motivation

• 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

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

downstream activity

The vision:Bacterial targeting

via membrane display

The vision:Inter-cellular activationvia Lux quorum-sensing

The vision:Intra-cellular activation

via Fec signal transduction

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

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

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

Direct Selection Talon and StreptactinMagnetic Beads

Indirect SelectionMACS Magnetic Beads And Antibodies

Streptactin Magnetic beads-bind strep2 (strep binding peptide)

or

Talon Magnetic beads-bind polyhistidine tag

2 Methods for selecting/enriching for surface engineered bacteria

MACS microbeads-binds Mouse IgG antibodies

Y Anti-Strep2 tag Mouse IgG antibody

Anti-his tag antibodyY

YY

YY

Labeled cellsretained

Unlabeled cellsWashed away

After magneticselection

Direct Selection using Magnetic Beads

Direct Selection using Magnetic Beads

Fusion at C terminus: Bead Assay (His tag)

Beads

Pre-assay plates

Loop Insertion

• PCR product digestion & ligation – Primer design– Digest-Ligate-Transform motif

• Gene design– Insertion points created for inserting synthetic

constructs

Loop Insertion: PCR products

E S

Pre-loop1 OmpA

OmpA Portion with Modified loop1

E P

E|P digested vector

X P

Complete Plasmid

M

PCR productsLane1: Ladder

Lane2: 1st portion OmpA

Lane 3: strep2-OmpA portion 2

Lane 4: 6xHis-OmpA portion 2

PCR: final plasmid as a template

Red line indicates the 1 kb band

Gene Design

E PX SN

Gene Design: Operations

N P

X PM

M

Gene Design: Operations

N NX S

M

M

M

M

His/strep tags OR randomers

PCR Results

Red line indicates the 1 kb line (7/8)

Cell-Cell Signalling

Receiver

Sender

R

OHHL

Target(bead)

Reporter

luxI/luxR Quorum Sensing

+

Cell-Cell Signaling:Constructs

SenderReceiver

Single Cell Construct – “JT”

Two Cell Construct

ReceiverSender

Sharp increase in fluorescence indicates quorum activity

Amount of sender cells added

Fluorescence per cell

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

0

500

1000

1500

2000

2500

3000

Nondiluted JT 1in200 dilution T02 nondilute

Flu

ore

scen

ce o

ver

OD

0

20

40

60

80

100

120

140

160

180

200

220

240

After Overnight

Direct Magnetic Beads: Good Enrichment

Plate Drop Experiment with Enriched Sender

OmpA C-Terminus Library

5uL 50uL 200uL

Vector 2 33 lots

10mer 14 149 lots

15mer 6 275 lots

uL transformant vs. colony count

5uL of ligation reaction (4500 !! ng DNA) were transformed into chemically competent BL21 Gold DE3 Cells.

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

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

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.

Biobricking the Fec System

CONCLUSION

• To be added

ACKNOWLEDGEMENTS

• Thank you.