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 How does this fit into iGEM and standard parts paradigm? What are your parts? Modularity? Simplify, simplify, simplify. Reader’s digest version of results. Too many words, more hard-hitting, crisper. Use headings and explain verbally. Suggest a broader, real-world application. Potential targets. Increasing local concentration improves the efficacy/toxicity margin. We may have steered away from standard BioBricks, but we’re going for more general application. Talk more about applications, pictures

Potential Targets and Applications Bind Proteins Bind Toxins Bind Viruses Bind Tissue Bind DNA Bind Other Cells Bind Surface

Fec signal transduction Quorum-sensing Fec signal transduction Bacterial targeting Quorum-sensing Fec signal transduction Organizational slide, transition to first topic of bacterial targeting Quorum-sensing Fec signal transduction

Surface Engineered Bacteria Engineered to Bind and Signal Modified AIDA-1 Fusion Protein OmpA – C terminal insertion OmpA-Loop1 insertion AIDA-1 – N terminal insertion FecA – loop insertion Membrane Protein Add a slide about the concept of the library. 5

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 Cell Sorting with his and strep2 Combine all legends and get rid of borders, blow up figures BIGGERRRRR Insert as excel

After Separation <NEW TITLE> Test: Cell Sorting with AIDA1 + sender constructs (with his and strep2) Before Separation strep2 his Combine all legends and get rid of borders, blow up figures BIGGERRRRR

Results: (MORE DESCRIPTIVE TITLE HERE) Initial cultures Enriched cultures through selection Have captions, more explanation of what’s going on Successful selection of E.coli expressing His or Strep2 on surface

Fec signal transduction Bacterial targeting Make grey darker Quorum-sensing Fec signal transduction

luxI/luxR Quorum Sensing Reporter R Receiver + OHHL Sender

Cell-Cell Signaling Constructs 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 Sender

Switch-like Quorum Response Receiver Sender

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 Sender Receiver

Fec signal transduction Bacterial targeting Too many words. Redraw diagram more simply or erase unnecessary parts. Maybe make an animation. Quorum-sensing Fec signal transduction

Motivation: Fec System Goal: Direct cell signaling Method: Re-engineer an existing signaling pathway Fec system: well-characterized substrate specific

Overview of Fec System Too many words. Redraw diagram more simply or erase unnecessary parts. Maybe make an animation. 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. Loops 7 & 8 Too many words. Point out areas on the structure more clearly. If there’re no results from collaboration, don’t mention it. Ferguson AD et al. “Structural Basis of Gating by the Outer Membrane Transporter FecA. Science 2002 Mar 1: 295(5560) 1715-9

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 Too many words. Redraw diagram more simply or erase unnecessary parts. Maybe make an animation. 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 Find Excel sheet for this graph

CONCLUSION Targeting: AIDA Intercellular signaling: QS Intracellular signaling: Fec Make a point of using surface expression and random libraries as a distinguishing factor.

Future Directions Targeting: AIDA Intercellular signaling: QS Intracellular signaling: Fec Make a point of using surface expression and random libraries as a distinguishing factor. 24

ACKNOWLEDGEMENTS Advisors Teaching Fellows Funding George Church Debra Auguste Jagesh V. Shah William Shih Pamela Silver Alain Viel Tamara Brenner Teaching Fellows Nicholas Guido Bill Senapedis Mike Strong Harris Wang Funding HHMI Harvard Provost Harvard Life Sciences Division Harvard School of Engineering and Applied Sciences Team picture

N terminus modification of AIDA1

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 Mistake here with AIDA1 – we did not make additions to the C terminus 30