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 Introduction 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 Introduction Selecting/enriching for surface engineered bacteria Direct Selection –Direct magnetic beads Indirect selection –MACS –FACS

Harvard iGEM 2007 Introduction Various Cell Sorting Experiments with his and strep2 - Assay Results Indirect Bead Assays Direct Bead Assays

Harvard iGEM 2007 Introduction Test: Cell Sorting with AIDA1 + sender constructs (with his and strep2) - Assay Results his strep2 Before Separation After Separation

Harvard iGEM 2007 Introduction Results: Cell Selection Assays are a Success! Selecting for surface engineered bacteria (such as his and strep2) is possible through either direct or indirect cell separation.

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 BBa_T9002 The plate-drop experiment BBa_S03623 – BBa_I13507 T9002 OHHL Receiver -> GFP S23I07 Red OHHL sender

Harvard iGEM 2007 Quorum Sensing Plate Drop Experiment with Enriched Sender

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

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 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)

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 Two 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 2007 Two 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 2007 Conclusion CONCLUSION To be added

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