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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. The motivation To develop a system for targeting bacteria to a specific substrate and effecting a cellular response. - PowerPoint PPT Presentation
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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
Harvard iGEM 2007Introduction
The motivationTo develop a system for targeting bacteria
to a specific substrate and effecting a cellular response
Harvard iGEM 2007Introduction
Bind Proteins
Bind Other Cells
Bind Tissue
Bind Surface
Bind DNA/RNA
Bind Viruses
Bind Toxins
Potential Targets and Applications
Harvard iGEM 2007Introduction
Quorum-sensing Fec signal transduction
Bacterial targeting
Quorum-sensing Fec signal transduction
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
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
Harvard iGEM 2007Introduction
His/Strep2 –tagged bacteria are enriched by MACS
(after MACS selection)
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.
Harvard iGEM 2007Quorum Sensing
Bacterial targeting
Fec signal transductionQuorum-sensing
Harvard iGEM 2007Quorum Sensing
luxI/luxR Quorum Sensing
Sender
+
R
OHHL
Receiver
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
Harvard iGEM 2007Quorum Sensing
Switch-like Quorum Response
Sender
Receiver
Harvard iGEM 2007Quorum Sensing
Selection with Magnetic Beads
Sender
AIDA-1 – N terminal insertion
Harvard iGEM 2007Quorum Sensing
60-fold Enrichment through Direct Magnetic Beads
Control: no beads Selection with streptactin beads
Harvard iGEM 2007Quorum Sensing
Enriched senders activate quorum response
Receiver Sender
Harvard iGEM 2007Fec signal transduction
Bacterial targeting
Quorum-sensing Fec signal transduction
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
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
Harvard iGEM 2007Fec signal transduction
Overview of Fec SystemFerric citrate
Loops 7 & 8
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
Harvard iGEM 2007Fec signal transduction
Wild-Type GFP ExpressionGFP Fluorescence Assays
10mM Sodium Citrate, AA93 Co-transformed
Constitutive GFP
Un-induced AA93
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time (hh:mm)
RF
Us
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
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
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
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)
Harvard iGEM 2007Bacterial Targeting
N terminus modification of AIDA1
MACS Results
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Cell Types
Co
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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
Harvard iGEM 2007Bacterial Targeting
CSR by gene design
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