Implementing Non-Native Quorum Sensing in E. coli Galen Gao - - PowerPoint PPT Presentation

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Implementing Non-Native Quorum Sensing in E. coli Galen Gao - - PowerPoint PPT Presentation

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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 The Impact of Diabetes According

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SLIDE 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

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SLIDE 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

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SLIDE 3

11/2/2014 Caltech IGEM 3

Diabetes

Proinsulin Intermediary signaling molecules Tyrosine kinase receptor Insulin

Beta Cell

Muscle Cell

Export via exocytosis

Glucose uptake via endocytosis & glycogen production Endopeptidases

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SLIDE 4

11/2/2014 4

A Synthetic Cell-Cell Communicator

Caltech IGEM

Ligand-exporting genes pTet

Response Regulator histidine kinase receptor

Export complex Post-translationally modified signaling peptide

Reporter (GFP)

pResponse

Cell 1 Cell 2

aTc

TetR

pConstitutive

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SLIDE 5

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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SLIDE 6

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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SLIDE 7

11/2/2014

3 Quorum Sensing Systems

Caltech IGEM

agrB agrC

mature signaling peptide

agrB agrD agrC agrA

agrA agrD prepeptide

  • agrBDCA
  • lamBDCA
  • fsrABC
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SLIDE 8

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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SLIDE 9

11/2/2014 9

Ligand Export Testing

Caltech IGEM

Ligand-exporting genes pTet

Response Regulator histidine kinase receptor

Export complex Post-translationally modified signaling peptide

Reporter (GFP)

pResponse

Cell 1 Cell 2

aTc

TetR

pConstitutive

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SLIDE 10

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

Chemiluminescent substrate

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SLIDE 11

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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SLIDE 12

11/2/2014 12

Western Blot – Cell Lysate

Caltech IGEM

Fsr lanes

0 nM 50 nM 150 nM 250 nM 350 nM 450 nM

30 kDa 25 kDa 5 kDa

fsrB Membrane Component 3xFLAG GBAP fsrB fsrB Membrane Component 3xFLAG GBAP fsrB 3xFLAG GBAP

  • r

3xFLAG

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11/2/2014 13

Western Blot – Supernatant

Caltech IGEM

Fsr lanes

0 nM 50 nM 150 nM 250 nM 350 nM 450 nM

30 kDa 25 kDa 5 kDa

fsrB Membrane Component 3xFLAG GBAP fsrB fsrB Membrane Component 3xFLAG GBAP fsrB 3xFLAG GBAP

  • r

3xFLAG

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SLIDE 14

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LC/MS - Background

Caltech IGEM

Sample Separation Column Mass Spectrometer Detector

Time Counts Time Counts m/z = 200 m/z = 300

Example Read-out

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SLIDE 15

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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LC/MS – lamBDCA System

Caltech IGEM

m/z = 577.3 m/z = 373.2 m/z = 345.2 m/z = 260.1 LamBDCA QS System 250nM aTc Induction

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SLIDE 17

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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LC/MS – fsrABC System

Caltech IGEM

fsrABC QS System 500nM aTc Induction All m/z channels

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SLIDE 19

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Conclusions – Ligand Export

Caltech IGEM

Ligand-exporting genes pTet

Response Regulator histidine kinase receptor

Export complex Post-translationally modified signaling peptide

Reporter (GFP)

pResponse

Cell 1 Cell 2

aTc

TetR

pConstitutive

  • agrBDCA
  • lamBDCA
  • fsrABC
  • agrBDCA
  • lamBDCA

 fsrABC

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SLIDE 20

11/2/2014 20

Ligand Reception Testing

Caltech IGEM

Ligand-exporting genes pTet

Response Regulator histidine kinase receptor

Export complex Post-translationally modified signaling peptide

Reporter (GFP)

pResponse

Cell 1 Cell 2

aTc

TetR

pConstitutive

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SLIDE 21

Scaffolds in Two Component Systems

11/2/2014 21 Caltech IGEM 11/2/2014 21

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SLIDE 22

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Testing Scaffold Protein

11/2/2014 22 Caltech IGEM

500 1000 1500 2000 2500 3000 0.001 0.01 0.1

GFP Fluorescence (AU) Concentration of Arabinose (%)

GFP Fluorescence for p1521 & p1523

p1521 (no scaffold) p1523 (scaffold)

p1521 p1523

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SLIDE 23

11/2/2014 23

Combinatorial Promoter Testing

Caltech IGEM

Ligand-exporting genes pTet

Response Regulator histidine kinase receptor

Export complex Post-translationally modified signaling peptide

Reporter (GFP)

pResponse

Cell 1 Cell 2

aTc

TetR

pConstitutive

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SLIDE 24

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 24 Caltech IGEM

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11/2/2014 Caltech IGEM 25

GFP Fluorescence (au)

pTet/pLac promoter acts as AND gate

11/2/2014 25 Caltech IGEM

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SLIDE 26

11/2/2014 Caltech IGEM 26

GFP Fluorescence (au)

pLac/pTet acts as asym-AND gate

11/2/2014 26 Caltech IGEM

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SLIDE 27

11/2/2014 Caltech IGEM 27

GFP Fluorescence (au)

pBAD/pTet acts as single-input gate

11/2/2014 27 Caltech IGEM

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SLIDE 28

11/2/2014 28

Conclusions

Caltech IGEM

Ligand-exporting genes pTet

Response Regulator histidine kinase receptor

Export complex Post-translationally modified signaling peptide

Reporter (GFP)

pResponse

aTc

TetR

pConstitutive

  • agrBDCA
  • lamBDCA
  • fsrABC
  • agrBDCA
  • lamBDCA

 fsrABC Positive Control Scaffold System successfully tested pTet/pLac AND pLac/pTet asym-AND pBAD/pTet single-input

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SLIDE 29

11/2/2014 Caltech IGEM 29

Additional Contributions

11/2/2014 29 Caltech IGEM

  • TXTL Characterization of Existing

Biobrick Promoters

  • Novel Safety Considerations
  • Participation in Collaborative Survey with

UVA iGEM Team

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SLIDE 30

11/2/2014 Caltech IGEM 30

What is TXTL?

11/2/2014 30 Caltech 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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SLIDE 31

11/2/2014 Caltech IGEM 31

Promoter TXTL Characterization

11/2/2014 31 Caltech 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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SLIDE 32

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 32 Caltech IGEM

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SLIDE 33
  • 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 33 Caltech IGEM

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SLIDE 34

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 34 Caltech IGEM

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SLIDE 35

11/2/2014 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

Caltech IGEM

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SLIDE 36

11/2/2014 36

Acknowledgements

Caltech IGEM

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SLIDE 37

11/2/2014 37

Questions?

Caltech IGEM