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. Surface Engineered Bacteria Engineered to Bind and Signal
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.

6. Surface Engineered Bacteria Engineered to Bind and Signal
Positive Signal
Background
AIDA-1 his or
AIDA-1 strep2
Sender LuxI RFP
Amp and Kan
Kan
Amp
Co-transform
signal

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

8. Direct Selection using Magnetic Beads
After magnetic
selection
Convert tables to graphs/charts

9. Direct Selection using Magnetic Beads
Convert tables to graphs/charts

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

11. Cell-Cell Signaling: luxI/luxR Quorum Sensing
Reporter
Target
(bead) R
Receiver +
OHHL
Sender

12. Cell-Cell Signaling: Constructs
Sender
Receiver
Single Cell Construct – “JT”
Two Cell Construct
Make a point that we used existing BioBricks successfully, and what parts we’ve built ourselves, bam.
Also, split slides such that we show construct & fluorescence curves (both for two cell and single cell constructs)
Voigt paper acknowledgement
luxpR constructs
Change blown-up pics to simpler
Why use single-cell?
Receiver
Sender

13. Sharp increase in fluorescence indicates quorum activity
Clarify what the assay was, possibly use a plot instead of bars.

14. Selection with Direct Magnetic Beads
Control: no selection
Experimental: Selection with beads

15. 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

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

17. Plate Drop Experiment with Enriched Sender

18. 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

19. 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):

20. 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)

21. 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

22. 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.

23. 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)

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

25. 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