Implementing Non-Native Quorum Sensing in E. coli2014.igem.org/files/presentation/Caltech_Championship.pdf · Implementing Non-Native Quorum Sensing in E. coli Galen Gao Yuanyuan

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

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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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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A Synthetic Cell-Cell Communicator

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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


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


• Response mechanism

– Organisms sense and respond to changes

• Histidine Kinase

– Membrane-bound

– Sensor

– Auto-phosphorylates

• Response Regulator

– Mediator

– Expression of target genes

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Two Component Systems

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Bretl D. J., Demetriadou C., Zahrt T. C., Microbiol. Mol. Biol. Rev.December 2011 vol. 75 no. 4566-582

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3 Quorum Sensing Systems

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agrB

agrC

mature signaling peptide

agrB agrD agrC agrA

agrA agrDprepeptide

• agrBDCA• lamBDCA• fsrABC

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3 Quorum Sensing Systems

FsrB

FsrA

FsrC

GBAP (Mature Peptide)

FsrA FsrB FsrC

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Ligand Export Testing

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Response Regulator

histid

ine

kin

ase

recep

tor

Export complex



Cell 1 Cell 2

aTc

TetRpConstitutive

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Western Blot - Background

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lamB

FLAG

Amp. Resist.

lamD

lamB

Flagged prepeptide

Flagged ligand

Anti-FLAG Antibody

Secondary Antibody

Horseradish Peroxidase

Chemiluminescentsubstrate

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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

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Western Blot – Cell Lysate

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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



3xFLAG GBAP fsrB 3xFLAG GBAPor

3xFLAG

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Western Blot – Supernatant

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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



3xFLAG GBAP fsrB 3xFLAG GBAPor

3xFLAG

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LC/MS - Background

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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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LC/MS – lam Expectations

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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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LC/MS – lamBDCA System

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m/z = 577.3

m/z = 373.2

m/z = 345.2

m/z = 260.1

LamBDCAQS System

250nM aTcInduction

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LC/MS – fsr Expectations

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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

http://dx.doi.org/10.1128/JB.00969-06

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LC/MS – fsrABC System

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fsrABCQS System

500nM aTcInduction

All m/z channels

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Conclusions – Ligand Export

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Response Regulator

histid

ine

kin

ase

recep

tor

Export complex



Cell 1 Cell 2

aTc

TetRpConstitutive


• agrBDCA• lamBDCA fsrABC

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Ligand Reception Testing

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Response Regulator

histid

ine

kin

ase

recep

tor

Export complex



Cell 1 Cell 2

aTc

TetRpConstitutive

Scaffolds in Two Component Systems

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Testing Scaffold Protein

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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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Combinatorial Promoter Testing

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Response Regulator

histid

ine

kin

ase

recep

tor

Export complex



Cell 1 Cell 2

aTc

TetRpConstitutive


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



GFP Fluorescence (au)

pTet/pLac promoter acts as AND gate




pLac/pTet acts as asym-AND gate




pBAD/pTet acts as single-input gate


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Conclusions

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Response Regulator

histid

ine

kin

ase

recep

tor

Export complex



aTc

TetRpConstitutive


• agrBDCA• lamBDCA fsrABC

Positive Control Scaffold System successfully tested

pTet/pLac ANDpLac/pTet asym-ANDpBAD/pTet single-input


Additional Contributions


• TXTL Characterization of Existing Biobrick Promoters

• Novel Safety Considerations

• Participation in Collaborative Survey with UVA iGEM Team


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


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

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


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


Drug Regulation and Testing


We contributed to the Synthetic Biology Global Awareness and Acceptance Survey Map spearheaded by the UVA iGEM Team.


Collaboration with UVA iGEM


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Acknowledgements

Victoria Hsiao & Anu ThubagereRichard MurrayMurray Biocircuits GroupYong WuNathan Dalleska

The Caltech SFP OfficeThe Amgen FoundationHoward Hughes Medical Institute

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Acknowledgements

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Questions?

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Documents

Implementing Non-Native Quorum Sensing in E. coli2014.igem.org/files/presentation/Caltech_Championship.pdf · Implementing Non-Native Quorum Sensing in E. coli Galen Gao Yuanyuan