cling-e. coli : bacteria on target harvard igem 2007 ellenor brown stephanie lo alex pickett sammy...
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Cling-E. coli :Bacteria on target
Harvard iGEM 2007Ellenor BrownStephanie LoAlex PickettSammy Sambu
Kevin SheePerry TsaiShaunak VankudreGeorge Xu
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The motivationTo develop a system for targeting bacteria
to a specific substrate and effecting a cellular response
Harvard iGEM 2007Introduction
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Bind Proteins
Bind Other Cells
Bind Tissue Bind Surface
Bind DNA/RNA
Bind VirusesBind Drugs/Toxins
Potential Targets and Applications
Harvard iGEM 2007Introduction
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Quorum-sensing Fec signal transduction
Bacterial targeting
Quorum-sensing Fec signal transduction
Harvard iGEM 2007Introduction
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Surface-engineered bacteria
Surface Protein
Membrane Protein
OmpA – C terminal insertion
OmpA-Loop1 insertion
AIDA-1 – N terminal insertion
Harvard iGEM 2007Bacterial Targeting
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Selecting/enriching forsurface-engineered bacteria
• Tags– Histidine tag + nickel– Strep2 tag + streptavidin
Magnetic Bead Assays
• Assay• Magnetic Activated Cell
Sorting (MACS)
Harvard iGEM 2007Bacterial Targeting
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Harvard iGEM 2007Bacterial Targeting
235 10 206 11
Colony counts after MACS selectionof tagged bacteria
His and Strep2 tagged bacteria bind to beads
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Harvard iGEM 2007Bacterial Targeting
Results
• Surface-expression vehicle – AIDA1• Engineered surface-displayed histidine tag and
strep2 tag• Demonstrated bacterial targeting to nickel and
streptavidin beads through MACS
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Bacterial targeting
Fec signal transductionQuorum-sensing
Harvard iGEM 2007Quorum Sensing
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luxI/luxR Quorum Sensing
Sender
+
R
OHHL
Receiver
Harvard iGEM 2007Quorum Sensing
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• 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 Constructs
Harvard iGEM 2007Quorum Sensing
Receiver
Sender
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Switch-like Quorum Response
Sender
ReceiverR
Harvard iGEM 2007Quorum Sensing
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MACS selection of cotransformedluxI-RFP/AIDA-strep2 cells
Harvard iGEM 2007Quorum Sensing
tetR luxI-RFP
Red sender
T7 AIDA-strep2
Strep2 tag
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Before After
Harvard iGEM 2007Quorum Sensing
MACS selection of cotransformedluxI-RFP/AIDA-strep2 cells
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Harvard iGEM 2007Quorum Sensing
luxI-RFP/AIDA-strep2 cells clump around streptavidin beads
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OHHL
Harvard iGEM 2007Quorum Sensing
Receiver
R
Selected luxI-RFP/AIDA-strep2 cells can send quorum signal
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Bacterial targeting
Quorum-sensing Fec signal transduction
Harvard iGEM 2007Fec signal transduction
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• Goal: Direct cell signaling• Method: Re-engineer an existing
signal transduction pathway• Fec system:
– well-characterized– only outer membrane signal transducer
Motivation: Fec System
Harvard iGEM 2007Fec signal transduction
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Overview of Fec SystemFerric citrate
Harvard iGEM 2007Fec signal transduction Braun et al. “Gene Regulation by Transmembrane Signaling.”
Biometals 2006 Apr;19(2):103-13.
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Ferric citrate
Loops 7 & 8
Loops 7 and 8 aspotential insertion sites
Harvard iGEM 2007Fec signal transduction 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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• From Braun lab (U. Tuebingen, Germany)– Fec knock-out strain, AA93– FecIRA plasmid– PFec-GFP plasmid
• pColA Duet Vector– Allows regulated expression of Fec genes under
T7 promoter
Constructs
Harvard iGEM 2007Fec signal transduction
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Fec-induced GFP expression
Harvard iGEM 2007Fec signal transduction
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• Problems:– Growth media: WL vs. LB?– Toxicity: membrane disruption?
• Goals:– Nickel and Streptavidin Binding– Finding new targets with signaling
• Random library• Computational Approach (Maranas lab)
Troubleshooting andNext Steps
Harvard iGEM 2007Fec signal transduction
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Bacterial targeting
Fec signaltransductionQuorum-sensing
Conclusions and Future Directions
Harvard iGEM 2007Harvard iGEM 2007Conclusion
Bacterial targeting
Fec signaltransductionQuorum-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 designOptimize localized quorum response
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AcknowledgementsAdvisors
George ChurchDebra AugusteJagesh V. Shah
William ShihPamela Silver
Alain VielTamara Brenner
Teaching FellowsNicholas GuidoBill SenapedisMike StrongHarris Wang
FundingHHMI
Harvard Provost
Harvard Life Sciences Division
Harvard School of Engineering and Applied Sciences
Special thanks to…Volkmar Braun
(U of Tuebingen)Costas Maranas
(Penn State U)
Harvard iGEM 2007Conclusion