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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 spatiallyspecific 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 OmpA – C terminal insertion Fusion Protein 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 Background Sender LuxI RFP Kan Co-transform Amp Amp and Kan Surface Engineered Bacteria Engineered to Bind and Signal Positive Signal AIDA-1 his or AIDA-1 strep2 Background Sender LuxI RFP Kan Amp and Kan Amp Co-transform signal 2 Methods for selecting/enriching for surface engineered bacteria Indirect Selection Direct Selection MACS Magnetic Beads And Antibodies Talon and Streptactin Magnetic Beads Streptactin Magnetic beads -bind strep2 (strep binding peptide) MACS microbeads -binds Mouse IgG antibodies or Talon Magnetic beads -bind polyhistidine tag Y Y Anti-Strep2 tag Mouse IgG antibody Anti-his tag antibody Labeled cells retained Unlabeled cells Washed away Y Y 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 Loop Insertion: PCR products Pre-loop1 OmpA E S X P E M OmpA Portion with Modified loop1 P Complete Plasmid E|P digested vector PCR products Lane1: 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 X N S P Gene Design: Operations N M X M P P Gene Design: Operations His/strep tags OR randomers X S N M M MN M PCR Results Red line indicates the 1 kb line (7/8) Cell-Cell Signalling luxI/luxR Quorum Sensing Reporter R Target (bead) Receiver + Sender OHHL Cell-Cell Signaling: Constructs Sender Single Cell Construct – “JT” Receiver Two Cell Construct Receiver 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 3000 0 20 40 60 80 100 120 140 160 180 200 220 240 After Overnight Fluorescence over OD 2500 2000 1500 1000 500 0 Nondiluted JT 1in200 dilution T02 nondilute 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 wellcharacterized 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 Transport 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 RFUs • 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. CONCLUSION • To be added ACKNOWLEDGEMENTS • Thank you.