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 AIDA-1 his or AIDA-1 strep2 Sender LuxI RFP Background Amp and Kan Kan Co-transform Amp

Surface Engineered Bacteria Engineered to Bind and Signal

Positive Signal AIDA-1 his or AIDA-1 strep2 Sender LuxI RFP Background Amp and Kan Kan Co-transform Amp signal

2 Methods for selecting/enriching for surface engineered bacteria Direct Selection

Talon and Streptactin Magnetic Beads

Indirect Selection

MACS Magnetic Beads And Antibodies

Streptactin Magnetic beads

-bind strep2 (strep binding peptide) or

Talon Magnetic beads

-bind polyhistidine tag

MACS microbeads -binds Mouse IgG antibodies Y Anti-Strep2 tag Mouse IgG antibody Y Anti-his tag antibody Y

Labeled cells retained Unlabeled cells Washed away

Direct Selection using Magnetic Beads After magnetic selection

Direct Selection using Magnetic Beads

Fusion at C terminus: Bead Assay (His tag) Pre-assay plates Beads

Loop Insertion

• • PCR product digestion & ligation – Primer design – Digest-Ligate-Transform motif Gene design – Insertion points created for inserting synthetic constructs

E X

Loop Insertion: PCR products

Pre-loop1 OmpA S OmpA Portion with Modified loop1 P E M P

PCR products

Lane1: Ladder Lane2: 1 st 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 X N S P

Gene Design: Operations

P P

X

Gene Design: Operations

His/strep tags OR randomers S M M N M M

PCR Results

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

Cell-Cell Signalling

luxI/luxR Quorum Sensing

Reporter Receiver + Sender Target (bead) R OHHL

Sender

Cell-Cell Signaling: Constructs

Single Cell Construct – “JT” Receiver Receiver Two Cell Construct 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 1500 1000 500 0 3000 2500 2000 Nondiluted JT 1in200 dilution T02 nondilute 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 Vector 2 10mer 14 15mer 6 50uL 33 149 275 200uL lots lots lots 5uL of ligation reaction (4500 !! ng DNA) were transformed into chemically competent BL21 Gold DE3 Cells.

uL transformant vs. colony count

• • • • 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

FecA Induction in Presence of Sodium Citrate in LB

• • • 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 2000 1500 1000 500 0 0:00:00 2:24:00 4:48:00 7:12:00

Time(mins)

9:36:00 12:00:00 14:24:00

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.

• To be added

CONCLUSION

ACKNOWLEDGEMENTS

• Thank you.