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Harvard iGEM 2007Introduction
Cling-E. coli :Bacteria on target
Harvard iGEM 2007Ellenor BrownStephanie LoAlex PickettSammy Sambu
Kevin SheePerry TsaiShaunak VankudreGeorge Xu
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Harvard iGEM 2007Introduction
The motivationTo develop a system for targeting bacteria
to a specific substrate and effecting a cellular response
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Harvard iGEM 2007Introduction
Bind Proteins
Bind Other Cells
Bind Tissue
Bind Surface
Bind DNA/RNA
Bind Viruses
Bind Toxins
Potential Targets and Applications
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Harvard iGEM 2007Introduction
Quorum-sensing Fec signal transduction
Bacterial targeting
Quorum-sensing Fec signal transduction
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Harvard iGEM 2007Introduction
Surface Engineered BacteriaEngineered to Bind and Signal
Fusion Protein
Membrane Protein
OmpA – C terminal insertion
OmpA-Loop1 insertion
AIDA-1 – N terminal insertion
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Harvard iGEM 2007Introduction
Selecting/enriching for surface engineered bacteria
• Tags– 6xHis + nickel beads– Strep2 + streptavidin
beads
• Assays– Magnetic Activated Cell
Sorting (MACS)– Fluorescence Activated
Cell Sorting (FACS)Fluorescence Bead Assays
Magnetic Bead Assays
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Harvard iGEM 2007Introduction
His/Strep2 –tagged bacteria are enriched by MACS
(after MACS selection)
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Harvard iGEM 2007Introduction
Results:
Cell Selection Assays are a Success! AIDA was re-engineered to target nickel and streptavidin with 6xHis and Strep2 tags
respectively, and selecting for surface-engineered bacteria was accomplished through magnetic activated cell sorting.
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Harvard iGEM 2007Quorum Sensing
Bacterial targeting
Fec signal transductionQuorum-sensing
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Harvard iGEM 2007Quorum Sensing
luxI/luxR Quorum Sensing
Sender
+
R
OHHL
Receiver
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Harvard iGEM 2007Quorum Sensing
• 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)
• Single Cell– Constitutive (Bba_J23039 + Bba_T9002)– Quorum Controlled (Bba_R0062 + Bba_A340620 + Bba_C0261 + Bba_E0240)
• Construction Intermediates
Cell-Cell Signaling ConstructsReceiver
Sender
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Harvard iGEM 2007Quorum Sensing
Switch-like Quorum Response
Sender
ReceiverR
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Harvard iGEM 2007Quorum Sensing
Selection with Magnetic Beads
Sender
AIDA-1 – N terminal insertion
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Harvard iGEM 2007Quorum Sensing
60-fold Selection through Magnetic Beads
Control: no beads Selection with streptavidin beads
Green (untagged)
Red (tagged)
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Harvard iGEM 2007Quorum Sensing
Enriched senders activate quorum response
Receiver Sender
OHHL
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Harvard iGEM 2007Fec signal transduction
Bacterial targeting
Quorum-sensing Fec signal transduction
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Harvard iGEM 2007Fec signal transduction
Motivation: Fec System
• Goal: Direct cell signaling
• Method: Re-engineer an existing signal transduction pathway
• Fec system:– well-characterized– substrate specific
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Harvard iGEM 2007Fec signal transduction
Overview of Fec System
Braun et al. “Gene Regulation by Transmembrane Signaling.” Biometals 2006 Apr;19(2):103-13.
Ferric citrate
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Harvard iGEM 2007Fec signal transduction
Overview of Fec SystemFerric citrate
Loops 7 & 8
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Harvard iGEM 2007Fec signal transduction
Constructs
• From Braun lab (U. Tuebingen, Germany)– Fec knock-out strain, AA93– FecIRA plasmid– Fec promoter, GFP plasmid
• pColA Duet Vector– Allows regulated expression of Fec genes
under T7 promoter
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Harvard iGEM 2007Fec signal transduction
Wild-Type GFP ExpressionGFP Fluorescence Assays
10mM Sodium Citrate, AA93 Co-transformed
Constitutive GFP
Un-induced AA93
0
200
400
600
800
1000
1200
1400
1600
1800
2000
0:00 2:24 4:48 7:12 9:36 12:00 14:24
time (hh:mm)
RF
Us
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Harvard iGEM 2007Fec signal transduction
Troubleshooting andNext Steps
• Problems:– Growth media– Toxicity: membrane disruption?
• Goals:– Nickel and Streptavidin Binding– Finding new targets with signaling
• Random library• Computational Approach
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Harvard iGEM 2007Conclusion
Conclusions• Targeting
– His and Strep2 tags on AIDA, targeting bacteria to nickel and streptavidin was successful
• Quorum sensing– Constructed one-cell system– Characterized two-cell system– Combined with targeting
• Fec signal transduction– Characterized Fec system
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Harvard iGEM 2007Conclusion
Future Directions• Bacterial targeting
– Trying out new targeting peptides (calmodulin)– Optimizing the random library approach in selecting for targeting
peptides
• Quorum sensing– Characterizing one-cell system– Optimizing quorum response after targeting
• Fec signal transduction– Effect signal transduction with targeting
• Application
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Harvard iGEM 2007Conclusion
AcknowledgementsAdvisors
George Church
Debra Auguste
Jagesh V. Shah
William Shih
Pamela Silver
Alain Viel
Tamara Brenner
Teaching FellowsNicholas Guido
Bill Senapedis
Mike Strong
Harris Wang
FundingHHMI
Harvard Provost
Harvard Life Sciences Division
Harvard School of Engineering and Applied Sciences
Special thanks to…Volkmar Braun
(University of Tuebingen)
Costas Maranas (Penn State University)
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Harvard iGEM 2007Bacterial Targeting
N terminus modification of AIDA1
MACS Results
white
white
red red 0
102030405060708090
100
aida1+strep2 aida1+his
Cell Types
Co
lon
y c
ou
nt
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Harvard iGEM 2007Bacterial Targeting
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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Harvard iGEM 2007Bacterial Targeting
CSR by gene design
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Harvard iGEM 2007Bacterial Targeting
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