implementing non-native quorum sensing in e....
TRANSCRIPT
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Implementing Non-Native Quorum Sensing in E. coli
Galen GaoYuanyuan (Tina) Xu
Bianca LepeMonique Alkiewicz
Anthony ChenAndrew Hou
Alec Lourenco
Mentors: Richard Murray, Victoria Hsiao, Anu Thubagere
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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
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11/2/2014 Caltech IGEM 3
Diabetes
Proinsulin
Intermediary signaling molecules
Ty
rosin
e kin
ase
recepto
r
Insulin
Beta Cell Muscle Cell
Export via exocytosis
Glucose uptake via endocytosis& glycogen production
Endopeptidases
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11/2/2014 4
A Synthetic Cell-Cell Communicator
Caltech IGEM
Ligand-exporting genes pTet
Response Regulator
histid
ine
kin
ase
recep
tor
Export complex
Post-translationallymodified signaling peptide
Reporter (GFP)pResponse
Cell 1 Cell 2
aTc
TetRpConstitutive
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11/2/2014 Caltech IGEM 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;68:4839-4849
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11/2/2014 Caltech IGEM 6
• Response mechanism
– Organisms sense and respond to changes
• Histidine Kinase
– Membrane-bound
– Sensor
– Auto-phosphorylates
• Response Regulator
– Mediator
– Expression of target genes
11/2/2014 6
Two Component Systems
Caltech IGEM
Bretl D. J., Demetriadou C., Zahrt T. C., Microbiol. Mol. Biol. Rev.December 2011 vol. 75 no. 4566-582
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11/2/2014
3 Quorum Sensing Systems
Caltech IGEM
agrB
agrC
mature signaling peptide
agrB agrD agrC agrA
agrA agrDprepeptide
• agrBDCA• lamBDCA• fsrABC
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11/2/2014 8
3 Quorum Sensing Systems
FsrB
FsrA
FsrC
GBAP (Mature Peptide)
FsrA FsrB FsrC
Caltech IGEM
• agrBDCA• lamBDCA• fsrABC
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11/2/2014 9
Ligand Export Testing
Caltech IGEM
Ligand-exporting genes pTet
Response Regulator
histid
ine
kin
ase
recep
tor
Export complex
Post-translationallymodified signaling peptide
Reporter (GFP)pResponse
Cell 1 Cell 2
aTc
TetRpConstitutive
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11/2/2014 10
Western Blot - Background
Caltech IGEM
lamB
FLAG
Amp. Resist.
lamD
lamB
Flagged prepeptide
Flagged ligand
Anti-FLAG Antibody
Secondary Antibody
Horseradish Peroxidase
Chemiluminescentsubstrate
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11/2/2014 11
fsrB Membrane Component 3xFLAG GBAP fsrB
fsrB Membrane Component 3xFLAG GBAP
fsrB Membrane Component
3xFLAG GBAP fsrB
3xFLAG GBAP
30.3 kDa
29 kDa
5.4 kDa
25 kDa
4 kDa
3xFLAG
FsrABC System
Western Blot – Potential Fragments
Caltech IGEM
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11/2/2014 12
Western Blot – Cell Lysate
Caltech IGEM
Fsr lanes
0 n
M
50
nM
150
nM
25
0 n
M
35
0 n
M
45
0 n
M
30 kDa
25 kDa
5 kDa
fsrB Membrane Component 3xFLAG GBAP fsrB
fsrB Membrane Component
3xFLAG GBAP fsrB 3xFLAG GBAPor
3xFLAG
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11/2/2014 13
Western Blot – Supernatant
Caltech IGEM
Fsr lanes
0 n
M
50
nM
150
nM
25
0 n
M
35
0 n
M
45
0 n
M
30 kDa
25 kDa
5 kDa
fsrB Membrane Component 3xFLAG GBAP fsrB
fsrB Membrane Component
3xFLAG GBAP fsrB 3xFLAG GBAPor
3xFLAG
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11/2/2014 14
LC/MS - Background
Caltech IGEM
Sample SeparationColumn
Mass Spectrometer
Detector
Time
Co
un
ts
Time
Co
un
ts
m/z = 200
m/z = 300
Example Read-out
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11/2/2014
LC/MS – lam Expectations
Caltech IGEM
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
15
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11/2/2014 16
LC/MS – lamBDCA System
Caltech IGEM
m/z = 577.3
m/z = 373.2
m/z = 345.2
m/z = 260.1
LamBDCAQS System
250nM aTcInduction
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11/2/2014
LC/MS – fsr Expectations
Caltech IGEM
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
15
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11/2/2014 18
LC/MS – fsrABC System
Caltech IGEM
fsrABCQS System
500nM aTcInduction
All m/z channels
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11/2/2014 19
Conclusions – Ligand Export
Caltech IGEM
Ligand-exporting genes pTet
Response Regulator
histid
ine
kin
ase
recep
tor
Export complex
Post-translationallymodified signaling peptide
Reporter (GFP)pResponse
Cell 1 Cell 2
aTc
TetRpConstitutive
• agrBDCA• lamBDCA• fsrABC
• agrBDCA• lamBDCA fsrABC
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11/2/2014 20
Ligand Reception Testing
Caltech IGEM
Ligand-exporting genes pTet
Response Regulator
histid
ine
kin
ase
recep
tor
Export complex
Post-translationallymodified signaling peptide
Reporter (GFP)pResponse
Cell 1 Cell 2
aTc
TetRpConstitutive
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Scaffolds in Two Component Systems
11/2/2014 21Caltech IGEM11/2/2014 21
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11/2/2014 22
Testing Scaffold Protein
11/2/2014 22Caltech IGEM
0
500
1000
1500
2000
2500
3000
0 0.001 0.01 0.1
GF
P F
luo
resc
ence
(A
U)
Concentration of Arabinose (%)
GFP Fluorescence for p1521 & p1523
p1521 (no scaffold) p1523 (scaffold)
p1521
p1523
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11/2/2014 23
Combinatorial Promoter Testing
Caltech IGEM
Ligand-exporting genes pTet
Response Regulator
histid
ine
kin
ase
recep
tor
Export complex
Post-translationallymodified signaling peptide
Reporter (GFP)pResponse
Cell 1 Cell 2
aTc
TetRpConstitutive
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11/2/2014 Caltech IGEM 24
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
Combinatorial Promoters
11/2/2014 24Caltech IGEM
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11/2/2014 Caltech IGEM 25
GFP Fluorescence (au)
pTet/pLac promoter acts as AND gate
11/2/2014 25Caltech IGEM
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11/2/2014 Caltech IGEM 26
GFP Fluorescence (au)
pLac/pTet acts as asym-AND gate
11/2/2014 26Caltech IGEM
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11/2/2014 Caltech IGEM 27
GFP Fluorescence (au)
pBAD/pTet acts as single-input gate
11/2/2014 27Caltech IGEM
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11/2/2014 28
Conclusions
Caltech IGEM
Ligand-exporting genes pTet
Response Regulator
histid
ine
kin
ase
recep
tor
Export complex
Post-translationallymodified signaling peptide
Reporter (GFP)pResponse
aTc
TetRpConstitutive
• agrBDCA• lamBDCA• fsrABC
• agrBDCA• lamBDCA fsrABC
Positive Control Scaffold System successfully tested
pTet/pLac ANDpLac/pTet asym-ANDpBAD/pTet single-input
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11/2/2014 Caltech IGEM 29
Additional Contributions
11/2/2014 29Caltech IGEM
• TXTL Characterization of Existing Biobrick Promoters
• Novel Safety Considerations
• Participation in Collaborative Survey with UVA iGEM Team
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11/2/2014 Caltech IGEM 30
What is TXTL?
11/2/2014 30Caltech IGEM
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
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11/2/2014 Caltech IGEM 31
Promoter TXTL Characterization
11/2/2014 31Caltech IGEM
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
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How feasible would it be to test and distribute a drug that can treat diabetes in animals and humans?
11/2/2014 Caltech IGEM 32
Drug Regulation and Testing
11/2/2014 32Caltech IGEM
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• 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 Caltech IGEM 33
Drug Regulation and Testing
11/2/2014 33Caltech IGEM
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We contributed to the Synthetic Biology Global Awareness and Acceptance Survey Map spearheaded by the UVA iGEM Team.
11/2/2014 Caltech IGEM 34
Collaboration with UVA iGEM
11/2/2014 34Caltech IGEM
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11/2/2014 35
Acknowledgements
Victoria Hsiao & Anu ThubagereRichard MurrayMurray Biocircuits GroupYong WuNathan Dalleska
The Caltech SFP OfficeThe Amgen FoundationHoward Hughes Medical Institute
Caltech IGEM
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11/2/2014 36
Acknowledgements
Caltech IGEM
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11/2/2014 37
Questions?
Caltech IGEM