Implementing Non-Native Quorum Sensing in E. coli Galen Gao Yuanyuan (Tina) Xu Bianca Lepe Monique Alkiewicz Anthony Chen Andrew Hou Alec Lourenco Mentors: Richard Murray, Victoria Hsiao, Anu Thubagere
The Impact of Diabetes • According to The American Diabetes Association, in 2012, over 29 million Americans were affected by diabetes. • Total cost of diagnosed diabetes in the United States in 2012: $245 billion • A rises as a consequence of the body’s inability to regulate blood glucose levels. • There are 2 forms of diabetes: Type I: Inability to produce insulin Type II: Cells not responsive to insulin
Diabetes Insulin receptor Tyrosine kinase Export via exocytosis Intermediary signaling molecules Proinsulin Endopeptidases Glucose uptake via endocytosis & glycogen production Beta Cell Muscle Cell 11/2/2014 Caltech IGEM 3
A Synthetic Cell-Cell Communicator Post-translationally modified signaling peptide receptor histidine kinase Export complex TetR pConstitutive Response Regulator aTc Ligand-exporting genes pTet Reporter (GFP) pResponse Cell 1 Cell 2 11/2/2014 Caltech IGEM 4
Quorum Sensing • A gene regulation system correlated with bacterial population size/density • Used to coordinate gene expression among large populations of bacteria • Bacteria secrete signaling molecules detected by receptors on other bacteria, affecting gene expression. de Kievit T R , and Iglewski B H Infect. Immun. 2000;68:4839-4849 11/2/2014 Caltech IGEM 5
Two Component Systems • Response mechanism – Organisms sense and respond to changes • Histidine Kinase – Membrane-bound – Sensor – Auto-phosphorylates • Response Regulator – Mediator – Expression of target genes Bretl D. J., Demetriadou C., Zahrt T. C., Microbiol. Mol. Biol. Rev.December 2011 vol. 75 no. 4566-582 11/2/2014 11/2/2014 Caltech IGEM 6 6 Caltech IGEM
3 Quorum Sensing Systems • agrBDCA mature signaling peptide • lamBDCA • fsrABC agrB agr C agrD agrA prepeptide agrB agrD agrC agrA 11/2/2014 Caltech IGEM
3 Quorum Sensing Systems • agrBDCA GBAP (Mature Peptide) • lamBDCA • fsrABC FsrC FsrB FsrA FsrA FsrB FsrC 11/2/2014 Caltech IGEM 8
Ligand Export Testing Post-translationally modified signaling peptide receptor histidine kinase Export complex TetR pConstitutive Response Regulator aTc Ligand-exporting genes pTet Reporter (GFP) pResponse Cell 1 Cell 2 11/2/2014 Caltech IGEM 9
Western Blot - Background Secondary Antibody Anti-FLAG Antibody Horseradish Peroxidase Flagged ligand lamB Chemiluminescent substrate lamB FLAG lamD Flagged prepeptide Amp. Resist. 11/2/2014 Caltech IGEM 10
Western Blot – Potential Fragments FsrABC System fsrB Membrane Component 3xFLAG GBAP fsrB 30.3 kDa fsrB Membrane Component 3xFLAG GBAP 29 kDa fsrB Membrane Component 3xFLAG 25 kDa 3xFLAG GBAP fsrB 5.4 kDa 3xFLAG GBAP 4 kDa 11/2/2014 Caltech IGEM 11
Western Blot – Cell Lysate Fsr lanes 0 nM 50 nM 250 nM 350 nM 450 nM 150 nM fsrB Membrane Component 3xFLAG GBAP fsrB 30 kDa 25 kDa fsrB Membrane Component 3xFLAG 5 kDa or 3xFLAG GBAP fsrB 3xFLAG GBAP 11/2/2014 Caltech IGEM 12
Western Blot – Supernatant Fsr lanes 0 nM 50 nM 250 nM 350 nM 450 nM 150 nM 30 kDa fsrB Membrane Component 3xFLAG GBAP fsrB 25 kDa fsrB Membrane Component 3xFLAG 5 kDa or 3xFLAG GBAP fsrB 3xFLAG GBAP 11/2/2014 Caltech IGEM 13
LC/MS - Background Separation Sample Column Mass Spectrometer Example Read-out m/z = 200 Counts Time m/z = 300 Counts Detector Time 11/2/2014 Caltech IGEM 14
LC/MS – lam Expectations Sturme, M.H.J., et al. (2005). An agr-Like Two-Component Regulatory System in Lactobacillus plantarum is Involved in Production of a Novel Cyclic Peptide and Regulation of Adherence . J. Bacteriol. , 187 , 5224-5235. doi:10.1128/JB.187.15.5224-5235 11/2/2014 Caltech IGEM 15
LC/MS – lamBDCA System m/z = 577.3 LamBDCA m/z = 373.2 QS System 250nM aTc Induction m/z = 345.2 m/z = 260.1 11/2/2014 Caltech IGEM 16
LC/MS – fsr Expectations Nakayama, J. et al. (2007). Siamycin Attenuates fsr Quorum Sensing Mediated by a Gelatinase Biosynthesis-Activating Pheromone in Enterococcus faecalis. J. Bacteriol. , 189 , 1358 – 1365. doi: 10.1128/JB.00969-06 11/2/2014 Caltech IGEM 15
LC/MS – fsrABC System fsrABC QS System All m/z channels 500nM aTc Induction 11/2/2014 Caltech IGEM 18
Conclusions – Ligand Export Post-translationally modified signaling peptide receptor histidine kinase • agrBDCA Export • lamBDCA complex fsrABC • TetR pConstitutive Response Regulator aTc Ligand-exporting genes pTet • agrBDCA Reporter (GFP) pResponse • lamBDCA fsrABC Cell 1 Cell 2 11/2/2014 Caltech IGEM 19
Ligand Reception Testing Post-translationally modified signaling peptide receptor histidine kinase Export complex TetR pConstitutive Response Regulator aTc Ligand-exporting genes pTet Reporter (GFP) pResponse Cell 1 Cell 2 11/2/2014 Caltech IGEM 20
Scaffolds in Two Component Systems 11/2/2014 11/2/2014 Caltech IGEM 21 21
Testing Scaffold Protein p1523 GFP Fluorescence for p1521 & p1523 3000 2500 GFP Fluorescence (AU) 2000 1500 p1521 1000 500 0 0 0.001 0.01 0.1 Concentration of Arabinose (%) p1521 (no scaffold) p1523 (scaffold) 11/2/2014 11/2/2014 Caltech IGEM 22 22
Combinatorial Promoter Testing Post-translationally modified signaling peptide receptor histidine kinase Export complex TetR pConstitutive Response Regulator aTc Ligand-exporting genes pTet Reporter (GFP) pResponse Cell 1 Cell 2 11/2/2014 Caltech IGEM 23
Combinatorial Promoters Cox, R. S., Surette, M. G., & Elowitz, M. B. (2007). Programming gene expression with combinatorial promoters. Molecular Systems Biology , 3 , 145 – 145. doi:10.1038/msb4100187 11/2/2014 11/2/2014 24 24 Caltech IGEM Caltech IGEM
pTet/pLac promoter acts as AND gate GFP Fluorescence (au) 11/2/2014 11/2/2014 25 25 Caltech IGEM Caltech IGEM
pLac/pTet acts as asym-AND gate GFP Fluorescence (au) 11/2/2014 11/2/2014 26 26 Caltech IGEM Caltech IGEM
pBAD/pTet acts as single-input gate GFP Fluorescence (au) 11/2/2014 11/2/2014 Caltech IGEM 27 27 Caltech IGEM
Conclusions Post-translationally modified signaling Positive Control peptide Scaffold System receptor histidine kinase successfully tested • agrBDCA Export • lamBDCA complex fsrABC • TetR pConstitutive Response Regulator aTc Ligand-exporting genes pTet • agrBDCA Reporter (GFP) pResponse • lamBDCA pTet/pLac AND fsrABC pLac/pTet asym-AND pBAD/pTet single-input 11/2/2014 Caltech IGEM 28
Additional Contributions • TXTL Characterization of Existing Biobrick Promoters • Novel Safety Considerations • Participation in Collaborative Survey with UVA iGEM Team 11/2/2014 11/2/2014 Caltech IGEM 29 29 Caltech IGEM
What is TXTL? TXTL is a cell-free transcription translation expression system that allows for rapid prototyping and debugging of biological circuits. Hong SH, Kwon YC, Jewett MC. Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis. Front Chem. 2014 Jun 10; 2:34 11/2/2014 11/2/2014 Caltech IGEM 30 30 Caltech IGEM
Promoter TXTL Characterization Measured expression levels of RFP under regulation by Anderson family of constitutive promoters (Berkeley iGEM 2006) in TX-TL * *in vivo values as reported in Biobrick registry 11/2/2014 11/2/2014 Caltech IGEM 31 31 Caltech IGEM
Drug Regulation and Testing How feasible would it be to test and distribute a drug that can treat diabetes in animals and humans? 11/2/2014 11/2/2014 Caltech IGEM 32 32 Caltech IGEM
Drug Regulation and Testing • Interviewed Kathleen Gilbert from Caltech Safety Office • IACUC Animal Testing Guidelines – Proper housing facilities – Standard protocol for care and management – Euthanasia of sick and dying animals • IRB Human Testing Guidelines – Protocol Application – Informed Consent Document • FDA Guidelines – Good Clinical Practices (GCPs) – Human Subject Protection (HSP) – Bioresearch Monitoring Program 11/2/2014 11/2/2014 Caltech IGEM 33 33 Caltech IGEM
Collaboration with UVA iGEM We contributed to the S ynthetic Biology Global Awareness and Acceptance Survey Map spearheaded by the UVA iGEM Team. 11/2/2014 11/2/2014 Caltech IGEM 34 34 Caltech IGEM
Acknowledgements Victoria Hsiao & Anu Thubagere Richard Murray Murray Biocircuits Group Yong Wu Nathan Dalleska The Caltech SFP Office The Amgen Foundation Howard Hughes Medical Institute 11/2/2014 Caltech IGEM 35
Acknowledgements 11/2/2014 Caltech IGEM 36
Questions? 11/2/2014 Caltech IGEM 37
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