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. 5

6. Selecting/enriching for surface engineered bacteria
Direct Selection
Direct magnetic beads
Indirect selection
MACS
FACS
Figure here to illustrate Direct selection and indirect selection

7. Cell Sorting with his and strep2
Indirect Bead Assays
Direct Bead Assays
Cell Sorting with his and strep2
Combine all legends and get rid of borders, blow up figures BIGGERRRRR
Insert as excel

8. After Separation
<NEW TITLE>
Test: Cell Sorting with AIDA1 + sender constructs (with his and strep2)
Before Separation
strep2
his
Combine all legends and get rid of borders, blow up figures BIGGERRRRR

9. Results: (MORE DESCRIPTIVE TITLE HERE)
Initial cultures
Enriched cultures through selection
Have captions, more explanation of what’s going on
Successful selection of E.coli expressing His or Strep2 on surface

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

11. luxI/luxR Quorum Sensing
Reporter R
Receiver +
OHHL
Sender

12. 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

13. Switch-like Quorum Response
Receiver
Sender

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

15. 60-fold Enrichment through Direct Magnetic Beads
Control: no beads
Selection with streptactin beads

16. The plate-drop experiment
Sender
Receiver

17. 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

18. 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):

19. 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)

20. 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

21. 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.

22. 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)

23. CONCLUSION Targeting: AIDA Intercellular signaling: QS
Intracellular signaling: Fec
Make a point of using surface expression and random libraries as a distinguishing factor.

24. Future Directions Targeting: AIDA Intercellular signaling: QS
Intracellular signaling: Fec
Make a point of using surface expression and random libraries as a distinguishing factor. 24

25. ACKNOWLEDGEMENTS Advisors Teaching Fellows Funding 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
Team picture

26. N terminus modification of AIDA1

27. 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

28. CSR by gene design

29. 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
<OmpA AIDA1 structures>
Fusion of tags & randomers to extracellular portion of OmpA loop 1 (loop insertion)
PCR product insertion (950 bps)
Insertion of ds oligos
Mistake here with AIDA1 – we did not make additions to the C terminus 30