towards intelligent probiotics chris brasseaux, ee david golynskiy, bio/crim tyler guinn, biochem/ee...
TRANSCRIPT
![Page 1: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/1.jpg)
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)
![Page 2: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/2.jpg)
Towards Intelligent Probiotics
1. Introduction
2. Immunobot: sensor-taxis
3. Killbot: population control
4. More
![Page 3: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/3.jpg)
Towards Intelligent Probiotics
1. Introduction
2. Immunobot: sensor-taxis
3. Killbot: population control
4. More
![Page 4: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/4.jpg)
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
![Page 5: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/5.jpg)
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
![Page 6: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/6.jpg)
Towards Intelligent Probiotics
• “Device 1”: Immunobot– Immune interface– Drug delivery
• “Device 2”: Killbot– System control
![Page 7: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/7.jpg)
Towards Intelligent Probiotics
1. Introduction
2. Immunobot: sensor-taxis
3. Killbot: population control
4. More
![Page 8: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/8.jpg)
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
![Page 9: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/9.jpg)
FGF Receptor (FGFR)
![Page 10: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/10.jpg)
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
![Page 11: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/11.jpg)
Connection to Chemotaxis
![Page 12: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/12.jpg)
Immunobot System
![Page 13: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/13.jpg)
Cloning…
![Page 14: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/14.jpg)
More Cloning
![Page 15: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/15.jpg)
…and more cloning
![Page 16: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/16.jpg)
Experiments
1. Transformed E.Coli Dh5a2. Incubated with nitrates for production of the receptor3. Introduced heparin and FGF 4. Performed fluorescent microscopy measurements
![Page 17: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/17.jpg)
Experiments
Negative Control
10uM heparin
100uM heparin
1mM heparin
15nM FGF220
225
230
235
240
245
250
255
260
PyeaR_FGFR-ToxR and ctx_GFP after 1 hourM
ean
Fluo
resc
ence
![Page 18: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/18.jpg)
Experiments
Negative Control
10uM heparin
100uM heparin
1mM heparin
15nM FGF210
215
220
225
230
235
240
245
250
255
PyeaR_FGFR-ToxR and ctx_GFP after 4 hoursM
ean
Fluo
resc
ence
![Page 19: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/19.jpg)
Experiments
no induc-tion
1uM heparin
10uM heparin
100uM heparin
1mM heparin
15nM FGF 30nM FGF210
215
220
225
230
235SCP-ToxR-FGFR and ctx-GFP after 1hour
Mea
n Fl
uore
scen
ce
![Page 20: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/20.jpg)
Interface to Chemotaxis
![Page 21: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/21.jpg)
Chemotaxis
Image from Roland Institute at Harvard Image from Roland McGraw Hill
![Page 22: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/22.jpg)
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 receptorModule 2: Transcription/translation of CheZModule 3: CheY dephosphorylation 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 phosphorylated CheY (Cluzel, Surette et al. 2000).
![Page 23: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/23.jpg)
Towards Intelligent Probiotics
1. Introduction
2. Immunobot: sensor-taxis
3. Killbot: population control
4. More
![Page 24: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/24.jpg)
Killbot: A suicide mechanism
This mechanism uses two plasmids:
1. PcstA*-RBS-LuxI-double terminator (Berkeley 2006)
2. AHL Inducible Colicin E2 with GFP (Calgary 2008)
![Page 25: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/25.jpg)
A Glucose Repressible Killbot
Time
Del
iver
y/G
luco
se
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Killbot Experiments
Population 1 (Immunobot) Population 1 (Immunobot) + more Population 2 (Killbot)
Population 1 (Immunobot) + Population 2 (Killbot)
• BL21 colicin sensitive cells (used same OD)• Population 1: AHL inducible colicin E2 with GFP• Population 2: glucose-repressible AHL producer
![Page 27: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/27.jpg)
-1.66533453693773E-16
0.2
0.4
0.6
0.8
1
1.2
Inte
grat
ed F
luor
esce
nt In
tens
ity
Killbot Experiments
The killbots eliminate the majority of the cells
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-1.66533453693773E-16
0.2
0.4
0.6
0.8
1
1.2
Inte
grat
ed F
luor
esce
nt In
tens
ity
Killbot Experiments
The addition of glucose in the medium increases the population two-fold
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Towards Intelligent Probiotics
1. Introduction
2. Immunobot: sensor-taxis
3. Killbot: population control
4. More
![Page 30: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/30.jpg)
Android Apps
![Page 31: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/31.jpg)
Android Apps
![Page 32: Towards Intelligent Probiotics Chris Brasseaux, EE David Golynskiy, Bio/Crim Tyler Guinn, BioChem/EE Sameer Sant, Bio/Econ Mitu Bhattatiry, Biomed (Columbia)](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649c855503460f9493ac5f/html5/thumbnails/32.jpg)
Biobricks
Name Type Description Designer Length
BBa_K569001 Composite PcstA+RBS+LuxI+double terminator Mitu Bhattatiry 937
BBa_K569013 Composite PyeaR+ToxR+FGFR David Golynskiy & Tyler Guinn 1536
BBa_K569014 Composite SCP+ToxR+FGFR David Golynskiy & Tyler Guinn 1471
Name Type Description Designer Length
BBa_K569000 Intermediate RBS+LuxI+dblterm Mitu Bhattatiry 798
BBa_K569003 Composite Phototaxis Receptor+eYFP Jose Alfredo Flores 2918
BBa_K569004 Composite Phototaxis Receptor+GFP Jose Alfredo Flores 2916
BBa_K569005 Composite Ctx-CheZ David Golynskiy & Tyler Guinn 790
BBa_K569005 Composite Ctx-CheZ David Golynskiy & Tyler Guinn 790
BBa_K569006 Composite Ctx-CheY David Golynskiy & Tyler Guinn 571
BBa_K569007 Coding CheZ mutant David Golynskiy & Tyler Guinn 664
BBa_K569010 Composite ctx+CheZ mutant David Golynskiy & Tyler Guinn 1701
BBa_K569011 Coding FGFR David Golynskiy & Tyler Guinn 792
BBa_K569012 Composite ToxR+FGFR David Golynskiy & Tyler Guinn 1430
BBa_K569017 Coding CheY David Golynskiy & Tyler Guinn 418
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Accomplishments
1. Built new BioBricks relating to wound sensing and chemotactic abilities
2. Demonstrated that some of them, particularly the killbot, seem to work as
expected.
3. Improved the characterization existing BioBricks, and included our
experience in the appropriate Registry page.
4. 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.
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Dr. Leonidas Bleris
Dr. Hyun-Joo Nam
Dr. Lan Ma
Neha Kashyap
Lagnajeet Pradhan
Kristina Ehrhardt