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Cling-E. coli : Bacteria on target

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1 Cling-E. coli : Bacteria on target
Try adding one single organizational slide to build upon as transitional slides between projects Harvard iGEM 2007 Ellenor Brown Stephanie Lo Alex Pickett Sammy Sambu Kevin Shee Perry Tsai Shaunak Vankudre George Xu

2 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

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

4 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

5 Cell Surface Reengineering (CSR)
CSR by PCR product digestion & ligation: Fusion of ds synthetic oligos to the C terminus of OmpA Fusion of ds synthetic oligos to the N terminus of AIDA1 Mistake here with AIDA1 – we did not make additions to the C terminus

6 N terminus modification of AIDA1

7 C terminus modification of OmpA
Pre-assay plates C terminus addition of his to the ompA gene The top shows the plates prior to separation by cobalt beads Bottom is the beads – whish have the bact containing the ompAhis + sender (with RFP) The ratios (1:100) are the dilutions I made prior to the assay – so there is in fact enrichment. Post-Assay Plates

8 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

9 CSR by gene design

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

11 Various Cell Sorting Experiments with his and strep2 -
Indirect Bead Assays Various Cell Sorting Experiments with his and strep2 - Assay Results Direct Bead Assays

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

13 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.

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

15 luxI/luxR Quorum Sensing
Reporter R Receiver + OHHL Sender

16 Cell-Cell Signaling Constructs
Receiver 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 Sender

17 Switch-like Quorum Response
Receiver Sender

18 Selection with Direct Magnetic Beads
Control: no selection Experimental: Selection with beads

19 60-fold Enrichment through Direct Magnetic Beads
Change “Experimental” to a more informative label & make the legend larger. Control: no beads Selection with streptactin beads

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

21 Plate Drop Experiment with Enriched Sender

22 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

23 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):

24 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. 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)

25 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 To be resulted

26 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. Conceptually good, but needs fewer words.

27 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. Studies detailing the response of reporter expression to mutations at each site of the promoter have been done. (Enz, Pressler, Braun papers)

28 CONCLUSION To be added Make a point of using surface expression and random libraries as a distinguishing factor.

29 ACKNOWLEDGEMENTS Advisors Teaching Fellows 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

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