Cling-E. coli : Bacteria on target

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Cling-E. coli Bacteria on target

• Harvard iGEM 2007

Kevin Shee Perry Tsai Shaunak Vankudre George Xu
Ellenor Brown Stephanie Lo Alex Pickett Sammy
Sambu
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The motivationTo develop a system for directing
bacteria to a target of interest and effecting
downstream activity
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Potential Targets and Applications
Bind Proteins
Bind Toxins
Bind Viruses
Bind Tissue
Bind DNA
Bind Other Cells
Bind Surface
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Bacterial targeting
Quorum-sensing
Fec signal transduction
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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
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Selecting/enriching for surface engineered
bacteria

• Direct Selection
• Direct magnetic beads
• Indirect selection
• MACS
• FACS

Figure here to illustrate Direct selection and
indirect selection
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Indirect Bead Assays
Direct Bead Assays
Cell Sorting with his and strep2
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After Separation
ltNEW TITLEgt Test Cell Sorting with AIDA1
sender constructs (with his and strep2)
Before Separation
strep2
his
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Results (MORE DESCRIPTIVE TITLE HERE)
Initial cultures
Enriched cultures through selection
Successful selection of E.coli expressing His or
Strep2 on surface
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Bacterial targeting
Fec signal transduction
Quorum-sensing
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luxI/luxR Quorum Sensing
Receiver

Sender
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Cell-Cell Signaling Constructs

• Receivers (luxR Reporter)
• GFP Receivers
• tetR controlled (Bba_T9002)
• Quorum controlled (Bba_R0062 Bba_C0261
  Bba_E0240)
• mRFP Receivers
• tetR controlled (Bba_F2620 Bba_I13507)
• Quorum controlled (Bba_R0062 Bba_C0261
  Bba_I13507)
• mCherry Receivers (Bba_F2620 Bba_J06702)
• Senders (bicistronic luxI Reporter)
• mRFP Sender
• tetR controlled (Bba_S03623 Bba_I13507)
• lacI controlled (Bba_S03608 Bba_I13507)
• Quorum controlled (Bba_R0062 Bba_A340620
  Bba_I13507)
• GFP Sender
• tetR controlled (Bba_S03623 Bba_E0240)
• lacI controlled (Bba_S03608 Bba_E0240)
• Quorum controlled (Bba_R0062 Bba_A340620
  Bba_E0240)
• mCherry Sender
• tetR controlled (Bba_S03623 Bba_J06702)

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Switch-like Quorum Response
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Selection with Direct Magnetic Beads
Control no selection
Experimental Selection with beads
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60-fold Enrichment through Direct Magnetic Beads
Control no beads
Selection with streptactin beads
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The plate-drop experiment
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Bacterial targeting
Quorum-sensing
Fec signal transduction
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Motivation Fec System

• Goal Direct cell signaling
• Method Re-engineer an existing signaling
  pathway
• Fec system
• well-characterized
• substrate specific

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Overview of Fec System
Braun et al. Gene Regulation by Transmembrane
Signaling. Biometals 2006 Apr19(2)103-13.
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Fec Motivation and Methods

• Re-engineer FecA Mutate loop 7 and/or loop 8
• Structural Evidence
• L7 moves up to 11Å, helix unwinds
• L8 moves up to 15Å
• Assume signaling will occur with binding.

Loops 7 8
Ferguson AD et al. Structural Basis of Gating by
the Outer Membrane Transporter FecA. Science
2002 Mar 1 295(5560) 1715-9
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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 Apr19(2)103-13.
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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

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CONCLUSION

• Targeting AIDA
• Intercellular signaling QS
• Intracellular signaling Fec

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Future Directions

• Targeting AIDA
• Intercellular signaling QS
• Intracellular signaling Fec

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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
Funding HHMI Harvard Provost Harvard Life
Sciences Division Harvard School of Engineering
and Applied Sciences
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N terminus modification of AIDA1
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C terminus modification of OmpA
Pre-assay plates
Post-Assay Plates
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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

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CSR by gene design
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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
• ltOmpA AIDA1 structuresgt
• Fusion of tags randomers to extracellular
  portion of OmpA loop 1 (loop insertion)
• PCR product insertion (950 bps)
• Insertion of ds oligos