Implementing Non-Native Quorum Sensing in E. coli

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1 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

2 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 Arises 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

3 Diabetes Insulin Export via exocytosis Tyrosine kinase receptor Intermediary signaling molecules Proinsulin Endopeptidases Glucose uptake via endocytosis & glycogen production Beta Cell Muscle Cell 3

4 A Synthetic Cell-Cell Communicator Post-translationally modified signaling peptide Export complex histidine kinase receptor pconstitutive atc TetR Response Regulator ptet Ligand-exporting genes presponse Reporter (GFP) Cell 1 Cell 2 4

5 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:

6 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 IGEM 6

7 3 Quorum Sensing Systems agrbdca lambdca fsrabc agrc mature signaling peptide agrb agra agrd prepeptide agrb agrd agrc agra 11/2/2014 Caltech IGEM

8 3 Quorum Sensing Systems agrbdca lambdca fsrabc GBAP (Mature Peptide) FsrC FsrB FsrA FsrA FsrB FsrC 8

9 Ligand Export Testing Post-translationally modified signaling peptide Export complex histidine kinase receptor pconstitutive atc TetR Response Regulator ptet Ligand-exporting genes presponse Reporter (GFP) Cell 1 Cell 2 9

10 Western Blot - Background Horseradish Peroxidase Secondary Antibody Anti-FLAG Antibody Flagged ligand lamb FLAG Chemiluminescent substrate lamb lamd Amp. Resist. Flagged prepeptide 10

11 Western Blot Potential Fragments FsrABC System 30.3 kda fsrb Membrane Component 3xFLAG GBAP fsrb 29 kda fsrb Membrane Component 3xFLAG GBAP 25 kda fsrb Membrane Component 3xFLAG 5.4 kda 3xFLAG GBAP fsrb 4 kda 3xFLAG GBAP 11

12 0 nm 50 nm 150 nm 250 nm 350 nm 450 nm Western Blot Cell Lysate Fsr lanes 30 kda 25 kda 5 kda fsrb Membrane Component 3xFLAG GBAP fsrb fsrb Membrane Component 3xFLAG 3xFLAG GBAP fsrb or 3xFLAG GBAP 12

13 0 nm 50 nm 150 nm 250 nm 350 nm 450 nm Western Blot Supernatant Fsr lanes 30 kda 25 kda fsrb Membrane Component 3xFLAG GBAP fsrb fsrb Membrane Component 3xFLAG 5 kda 3xFLAG GBAP fsrb or 3xFLAG GBAP 13

14 Counts Counts LC/MS - Background Sample Separation Column Example Read-out Mass Spectrometer m/z = 200 Time m/z = 300 Detector Time 14

15 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, doi: /jb /2/2014 Caltech IGEM 15

16 LC/MS lambdca System m/z = LamBDCA QS System 250nM atc Induction m/z = m/z = m/z =

17 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, doi: /JB /2/2014 Caltech IGEM 15

18 LC/MS fsrabc System fsrabc QS System 500nM atc Induction All m/z channels 18

19 Conclusions Ligand Export Post-translationally modified signaling peptide pconstitutive atc TetR Export complex agrbdca lambdca fsrabc Response Regulator histidine kinase receptor ptet Ligand-exporting genes agrbdca lambdca fsrabc presponse Cell 1 Cell 2 Reporter (GFP) 19

20 Ligand Reception Testing Post-translationally modified signaling peptide Export complex histidine kinase receptor pconstitutive atc TetR Response Regulator ptet Ligand-exporting genes presponse Reporter (GFP) Cell 1 Cell 2 20

21 Scaffolds in Two Component Systems 21

22 GFP Fluorescence (AU) Testing Scaffold Protein p GFP Fluorescence for p1521 & p p Concentration of Arabinose (%) p1521 (no scaffold) p1523 (scaffold) 22

23 Combinatorial Promoter Testing Post-translationally modified signaling peptide Export complex histidine kinase receptor pconstitutive atc TetR Response Regulator ptet Ligand-exporting genes presponse Reporter (GFP) Cell 1 Cell 2 23

24 Combinatorial Promoters Cox, R. S., Surette, M. G., & Elowitz, M. B. (2007). Programming gene expression with combinatorial promoters. Molecular Systems Biology, 3, doi: /msb

25 ptet/plac promoter acts as AND gate GFP Fluorescence (au) 25

26 plac/ptet acts as asym-and gate GFP Fluorescence (au) 26

27 pbad/ptet acts as single-input gate GFP Fluorescence (au) 27

28 Conclusions Post-translationally modified signaling peptide Export complex agrbdca lambdca fsrabc Positive Control Scaffold System successfully tested histidine kinase receptor pconstitutive atc TetR Response Regulator ptet Ligand-exporting genes agrbdca lambdca fsrabc presponse Reporter (GFP) ptet/plac AND plac/ptet asym-and pbad/ptet single-input 28

29 Additional Contributions TXTL Characterization of Existing Biobrick Promoters Novel Safety Considerations Participation in Collaborative Survey with UVA igem Team 29

30 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 Jun 10; 2:34 30

31 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 31

32 Drug Regulation and Testing How feasible would it be to test and distribute a drug that can treat diabetes in animals and humans? 32

33 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 33

34 Collaboration with UVA igem We contributed to the Synthetic Biology Global Awareness and Acceptance Survey Map spearheaded by the UVA igem Team. 34

35 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 35

36 Acknowledgements 36

37 Questions? 37