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Towards Intelligent Probiotics

Towards Intelligent Probiotics. Chris Brasseaux , EE David Golynskiy , Bio/ Crim Tyler Guinn, BioChem /EE Sameer Sant , Bio/Econ Mitu Bhattatiry , Biomed (Columbia) Nimi Bhattatiry (High School) Jose Alfredo Flores (Monterey Mexico). Towards Intelligent Probiotics. Introduction

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Towards Intelligent Probiotics

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  1. Towards Intelligent Probiotics Chris Brasseaux, EE DavidGolynskiy, Bio/Crim Tyler Guinn, BioChem/EE SameerSant, Bio/Econ • MituBhattatiry, Biomed (Columbia) NimiBhattatiry (High School) Jose Alfredo Flores (Monterey Mexico)

  2. Towards Intelligent Probiotics • Introduction • Immunobot: sensor-taxis • Killbot: population control • More

  3. Towards Intelligent Probiotics • Introduction • Immunobot: sensor-taxis • Killbot: population control • More

  4. Towards Intelligent Probiotics • Live microorganisms that give benefit to host (e.g. live cultures in yogurt) • May be used as therapeutic tool • Address disorders afflicting the intestine • May result in problematic tissue damage

  5. Towards Intelligent Probiotics • Human bowel symbionts represent engineering platform • Intelligent probiotics are user-controlled and confer health benefits to host • Stanford 2009: regulated lymphocytes to control inflammation • Goals • Interface with immune system to produce localization at damage site • Enable population control

  6. Towards Intelligent Probiotics • “Device 1”: Immunobot • Immune interface • Drug delivery • “Device 2”: Killbot • System control

  7. Towards Intelligent Probiotics • Introduction • Immunobot: sensor-taxis • Killbot: population control • More

  8. Signal Detection • Fibroblasts play important role in wound healing • Fibroblast growth factors (FGFs) are paracrine heparin-binding proteins that trigger fibroblast differentiation • FGFs can represent wound signals

  9. FGF Receptor (FGFR)

  10. FGF Receptor Interface • Kolmer et al., 1994 achieved a similar effect with two constructs: • Chimeric receptor: maltose receptor fused to ToxR transcription factor • CTX cloned upstream of lacZ genes Image from Kolmer, 1994

  11. Connection to Chemotaxis

  12. Immunobot System

  13. Cloning…

  14. More Cloning

  15. …and more cloning

  16. Experiments 1. Transformed E.Coli Dh5a 2. Incubated with nitrates for production of the receptor 3. Introduced heparin and FGF 4. Performed fluorescent microscopy measurements

  17. Experiments

  18. Experiments

  19. Experiments

  20. Interface to Chemotaxis

  21. Chemotaxis Image from Roland Institute at Harvard Image from Roland McGraw Hill

  22. Modeling The signaling network from the input of external ligand signal to the output of the tumbling state of a E coli cell can be quantitatively described by a modular model. The model is formulated based on the law of mass action and Michaelis-Menten mechanism and contains four relatively independent modules.Module 1: Activation of ToxR receptor Module 2: Transcription/translation of CheZModule 3:CheYdephosphorylation by the CheZ proteinModule 4: The tumbling activity of E coli is characterized by the so-called “bias”, which is defined as the ratio of the time of directed movement and the total time. It is experimentally measured that the bias is a Hill function dependent on the concentration of phosphorylatedCheY (Cluzel, Surette et al. 2000).

  23. Towards Intelligent Probiotics • Introduction • Immunobot: sensor-taxis • Killbot: population control • More

  24. Killbot: A suicide mechanism • This mechanism uses two plasmids: • PcstA*-RBS-LuxI-double terminator (Berkeley 2006) • AHL Inducible Colicin E2 with GFP (Calgary 2008)

  25. A Glucose Repressible Killbot Delivery/Glucose Time

  26. Killbot Experiments • BL21 colicin sensitive cells (used same OD) • Population 1: AHL inducible colicinE2 with GFP • Population 2: glucose-repressible AHL producer Population 1 (Immunobot) Population 1 (Immunobot) + Population 2 (Killbot) Population 1 (Immunobot) + more Population 2 (Killbot)

  27. Killbot Experiments The killbots eliminate the majority of the cells

  28. Killbot Experiments The addition of glucose in the medium increases the population two-fold

  29. Towards Intelligent Probiotics • Introduction • Immunobot: sensor-taxis • Killbot: population control • More

  30. Android Apps

  31. Android Apps

  32. Biobricks

  33. Accomplishments Built new BioBricks relating to wound sensing and chemotactic abilities Demonstrated that some of them, particularly the killbot, seem to work as expected. Improved the characterization existing BioBricks, and included our experience in the appropriate Registry page. Qualifying for MIT will allow us to test the different promoter-receptor constructs with longer induction times, different ligand concentrations, and the chemotaxis experiments with controlled gradient settings.

  34. Dr. LeonidasBleris Dr. Hyun-Joo Nam Dr. Lan Ma NehaKashyap LagnajeetPradhan Kristina Ehrhardt

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