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

Harvard iGEM 2007 Introduction The motivation To develop a system for directing bacteria to a target of interest and effecting downstream activity

Harvard iGEM 2007 Introduction Bind Proteins Bind Other Cells Bind Tissue Bind Surface Bind DNA Bind Viruses Bind Toxins Potential Targets and Applications

Harvard iGEM 2007 Introduction Quorum-sensingFec signal transduction Bacterial targeting Quorum-sensingFec signal transduction

Harvard iGEM 2007 Bacterial Targeting Surface Engineered Bacteria Engineered 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 2007 Bacterial 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 2007 Bacterial Targeting Cell Sorting with his and strep2 Indirect Bead Assays Direct Bead Assays

Harvard iGEM 2007 Bacterial Targeting Test: Cell Sorting with AIDA1 + sender constructs (with his and strep2) his strep2 Before Separation After Separation

Harvard iGEM 2007 Bacterial Targeting Results: (MORE DESCRIPTIVE TITLE HERE) Successful selection of E.coli expressing His or Strep2 on surface Initial culturesEnriched cultures through selection

Harvard iGEM 2007 Quorum Sensing Bacterial targeting Fec signal transductionQuorum-sensing

Harvard iGEM 2007 Quorum Sensing luxI/luxR Quorum Sensing Receiver Sender + Reporter R OHHL

Harvard iGEM 2007 Quorum Sensing Receivers (luxR + Reporter) –GFP Receivers (Bba_T9002) –mRFP Receivers (Bba_F Bba_I13507) –mCherry Receivers (Bba_F Bba_J06702) Senders (bicistronic luxI + Reporter) –mRFP Sender tetR controlled (Bba_S Bba_I13507) lacI controlled (Bba_S Bba_I13507) –GFP Sender tetR controlled (Bba_S Bba_E0240) lacI controlled (Bba_S Bba_E0240) –mCherry Sender tetR controlled (Bba_S Bba_J06702) Single Cell –Constitutive (Bba_J Bba_T9002) –Quorum Controlled (Bba_R Bba_A Bba_C Bba_E0240) Construction Intermediates Cell-Cell Signaling Constructs Receiver Sender

Harvard iGEM 2007 Quorum Sensing Switch-like Quorum Response Sender Receiver

Harvard iGEM 2007 Quorum Sensing Selection with Direct Magnetic Beads Control: no selection Experimental: Selection with beads

Harvard iGEM 2007 Quorum Sensing 60-fold Enrichment through Direct Magnetic Beads Control: no beadsSelection with streptactin beads

Harvard iGEM 2007 Quorum Sensing The plate-drop experiment Receiver Sender

Harvard iGEM 2007 Two Component System Bacterial targeting Quorum-sensingFec signal transduction

Harvard iGEM 2007 Two Component System Motivation: Fec System Goal: Direct cell signaling Method: Re-engineer an existing signaling pathway Fec system: – well-characterized – substrate specific

Harvard iGEM 2007 Two Component System Overview of Fec System Braun et al. “Gene Regulation by Transmembrane Signaling.” Biometals 2006 Apr;19(2):

Harvard iGEM 2007 Two Component System 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. Ferguson AD et al. “Structural Basis of Gating by the Outer Membrane Transporter FecA. Science 2002 Mar 1: 295(5560) Loops 7 & 8

Harvard iGEM 2007 Two 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):

Harvard iGEM 2007 Two 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

Harvard iGEM 2007 Conclusion CONCLUSION Targeting: AIDA Intercellular signaling: QS Intracellular signaling: Fec

Harvard iGEM 2007 Conclusion Future Directions Targeting: AIDA Intercellular signaling: QS Intracellular signaling: Fec

Harvard iGEM 2007 Conclusion ACKNOWLEDGEMENTS Advisors 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

Harvard iGEM 2007 Bacterial Targeting N terminus modification of AIDA1

Harvard iGEM 2007 Bacterial Targeting C terminus modification of OmpA Post-Assay Plates Pre-assay plates

Harvard iGEM 2007 Bacterial 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 2007 Bacterial Targeting CSR by gene design

Harvard iGEM 2007 Bacterial 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 Fusion of tags & randomers to extracellular portion of OmpA loop 1 (loop insertion) – PCR product insertion (950 bps) – Insertion of ds oligos