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 targeting bacteria to a specific substrate and effecting a cellular response ID: 785230
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Slide1
Cling-
E. coli
:
Bacteria on target
Harvard
iGEM
2007
Ellenor BrownStephanie LoAlex PickettSammy Sambu
Kevin SheePerry TsaiShaunak VankudreGeorge Xu
Slide2The motivation
To develop a system for targeting bacteria to a specific substrate and effecting a cellular response
Harvard iGEM 2007
Introduction
Slide3Bind Proteins
Bind Other Cells
Bind Tissue
Bind Surface
Bind DNA/RNA
Bind Viruses
Bind
Drugs/Toxins
Potential Targets and Applications
Harvard iGEM 2007
Introduction
Slide4Quorum-sensing
Fec
signal transduction
Bacterial targeting
Quorum-sensing
Fec signal transduction
Harvard iGEM 2007
Introduction
Slide5Surface-engineered
bacteria
Surface
Protein
Membrane Protein
OmpA
– C terminal insertion
OmpA-Loop1 insertion
AIDA-1 – N terminal insertion
Harvard
iGEM
2007
Bacterial Targeting
Slide6Selecting/enriching
forsurface-engineered bacteria
TagsHistidine tag + nickelStrep2 tag +
streptavidin
Magnetic Bead Assays
Assay
Magnetic Activated Cell Sorting (MACS)
Harvard
iGEM
2007
Bacterial Targeting
Slide7Harvard
iGEM
2007
Bacterial Targeting
235 10
206
11
Colony counts after MACS selection
of tagged bacteria
His and Strep2 tagged bacteria bind to beads
Slide8Harvard
iGEM
2007
Bacterial Targeting
Results
Surface-expression vehicle – AIDA1
Engineered surface-displayed
histidine
tag and strep2 tag
Demonstrated bacterial targeting to nickel and
streptavidin
beads through MACS
Slide9Bacterial targeting
Fec
signal transduction
Quorum-sensing
Harvard iGEM 2007
Quorum Sensing
Slide10luxI/luxR Quorum Sensing
Sender
+
R
OHHL
Receiver
Harvard iGEM 2007
Quorum Sensing
Slide11Receivers
(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 SendertetR 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)
Single CellConstitutive (Bba_J23039 +
Bba_T9002)Quorum Controlled (Bba_R0062 +
Bba_A340620 + Bba_C0261 + Bba_E0240
)Construction Intermediates
Cell-Cell Signaling Constructs
Harvard iGEM 2007
Quorum Sensing
Receiver
Sender
Slide12Switch-like Quorum Response
Sender
Receiver
R
Harvard iGEM 2007
Quorum Sensing
Slide13MACS selection of
cotransformed
luxI-RFP/AIDA-strep2 cells
Harvard iGEM 2007
Quorum Sensing
tetR
luxI
-RFP
Red sender
T7
AIDA-strep2
Strep2 tag
Slide14Before
After
Harvard iGEM 2007
Quorum Sensing
MACS selection of
cotransformed
luxI
-RFP/AIDA-strep2 cells
Slide15Harvard iGEM 2007
Quorum Sensing
luxI
-RFP/AIDA-strep2 cells clump around
streptavidin
beads
Slide16OHHL
Harvard iGEM 2007
Quorum Sensing
Receiver
R
Selected
luxI
-RFP/AIDA-strep2
cells can send quorum signal
Slide17Bacterial targeting
Quorum-sensing
Fec signal transduction
Harvard
iGEM
2007
Fec
signal transduction
Slide18Goal: Direct cell signaling
Method: Re-engineer an existing signal transduction pathwayFec system: well-characterized only outer membrane signal transducer
Motivation: Fec System
Harvard
iGEM
2007
Fec signal transduction
Slide19Overview of
Fec
System
Ferric citrate
Harvard
iGEM
2007
Fec
signal transduction
Braun et al. “Gene Regulation by
Transmembrane
Signaling.”
Biometals
2006 Apr;19(2):103-13
.
Slide20Ferric citrate
Loops 7 & 8
Loops
7 and 8 as
potential insertion sites
Harvard
iGEM
2007
Fec
signal transduction
Ferguson AD, et al. Structural basis of gating by the outer membrane transporter
FecA
. Science 2002 Mar 1; 295(5560) 1715-9.
Slide21From Braun lab (U.
Tuebingen, Germany)Fec knock-out strain, AA93FecIRA plasmid
PFec-GFP plasmidpColA Duet VectorAllows regulated expression of Fec
genes under T7 promoterConstructs
Harvard
iGEM
2007
Fec signal transduction
Slide22Fec
-induced GFP expression
Harvard
iGEM 2007
Fec
signal transduction
Slide23Problems:
Growth media: WL vs. LB?Toxicity: membrane disruption?
Goals:Nickel and Streptavidin BindingFinding new targets with signalingRandom library
Computational Approach (Maranas lab)
Troubleshooting andNext Steps
Harvard
iGEM
2007
Fec signal transduction
Slide24Bacterial targeting
Fec
signal
transduction
Quorum-sensing
Conclusions and Future Directions
Harvard
iGEM
2007
Harvard iGEM 2007
Conclusion
Bacterial targeting
Fec
signal
transduction
Quorum-sensing
Surface engineering – AIDA1
Histidine
/Strep2 tags
MACS
One-cell/two-cell quorum systems
Characterized
Targeted quorum senders
Characterized wild-type
Fec
signaling
Random peptide libraries
Computational design
Optimize localized quorum response
Slide25Acknowledgements
Advisors
George ChurchDebra AugusteJagesh V. Shah
William ShihPamela SilverAlain Viel
Tamara Brenner
Teaching FellowsNicholas GuidoBill SenapedisMike Strong
Harris Wang
FundingHHMIHarvard ProvostHarvard Life Sciences DivisionHarvard School of Engineering and Applied Sciences
Special thanks to…Volkmar Braun (U of Tuebingen
)
Costas
Maranas
(Penn
State
U)
Harvard iGEM 2007
Conclusion