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Developing a system to target bacteria to specific substrates and elicit a cellular response. Potential applications include binding proteins, toxins, tissue, DNA/RNA, viruses, and other cells.
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Cling-E. coli :Bacteria on target Harvard iGEM 2007 Ellenor Brown Stephanie Lo Alex Pickett Sammy Sambu Kevin Shee Perry Tsai Shaunak Vankudre George Xu
The motivationTo develop a system for targeting bacteria to a specific substrate and effecting a cellular response
Potential Targets and Applications Bind Proteins Bind Toxins Bind Tissue Bind DNA/RNA Bind Viruses Bind Surface Bind Other Cells
Bacterial targeting Quorum-sensing Fec signal transduction Quorum-sensing Fec signal transduction
Fusion Protein OmpA – C terminal insertion Membrane Protein OmpA-Loop1 insertion AIDA-1 – N terminal insertion Surface Engineered Bacteria Engineered to Bind and Signal
Selecting/enriching for surface engineered bacteria Tags 6xHis + nickel beads Strep2 + streptavidin beads • Assay • Magnetic Activated Cell Sorting (MACS) Magnetic Bead Assays
His/Strep2 –tagged bacteria are enriched by MACS (after MACS selection) 235 10 206 11
Results: Cell Selection Assays are a Success! AIDA was re-engineered to target nickel and streptavidin with His and Strep2 tags respectively, and selecting for surface-engineered bacteria was accomplished through magnetic activated cell sorting.
Bacterial targeting Quorum-sensing Fec signal transduction
luxI/luxR Quorum Sensing Receiver + R OHHL Sender
Receiver Sender Cell-Cell Signaling Constructs • Receivers (luxR + Reporter) • GFPReceivers • tetR controlled (Bba_T9002) • Quorum controlled (Bba_R0062 + Bba_C0261 + Bba_E0240) • mRFPReceivers • tetR controlled (Bba_F2620 + Bba_I13507) • Quorum controlled (Bba_R0062 + Bba_C0261 +Bba_I13507) • mCherryReceivers (Bba_F2620 + Bba_J06702) • Senders (bicistronic luxI + Reporter) • mRFPSender • tetR controlled (Bba_S03623 + Bba_I13507) • lacI controlled (Bba_S03608 + Bba_I13507) • Quorum controlled (Bba_R0062 + Bba_A340620 + Bba_I13507) • GFPSender • tetR controlled (Bba_S03623 + Bba_E0240) • lacI controlled (Bba_S03608 + Bba_E0240) • Quorum controlled (Bba_R0062 + Bba_A340620 + Bba_E0240) • mCherrySender • 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
Receiver Sender Switch-like Quorum Response R
Selection with Magnetic Beads AIDA-1 – N terminal insertion Sender
60-fold Selection through Magnetic Beads Control: no beads Selection with streptavidin beads Green (untagged) Red (tagged)
Receiver Sender Enriched senders activate quorum response OHHL
Bacterial targeting Quorum-sensing Fec signal transduction
Motivation: Fec System • Goal: Direct cell signaling • Method: Re-engineer an existing signal transduction pathway • Fec system: • well-characterized • substrate specific
Overview of Fec System Ferric citrate Braun et al. “Gene Regulation by Transmembrane Signaling.” Biometals 2006 Apr;19(2):103-13.
Overview of Fec System Ferric citrate Loops 7 & 8
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
Troubleshooting andNext Steps • Problems: • Growth media • Toxicity: membrane disruption? • Goals: • Nickel and Streptavidin Binding • Finding new targets with signaling • Random library • Computational Approach
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
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
Acknowledgements Teaching Fellows Nicholas Guido Bill Senapedis Mike Strong Harris Wang Advisors George Church Debra Auguste Jagesh V. Shah William Shih Pamela Silver Alain Viel Tamara Brenner Funding HHMI 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)
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
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