Quorum Sensing Disruption through pvdQ Production2012.igem.org/files/presentation/HKU_HongKong.pdf · 2012-10-06 · 1. Characterize the AHL inducible pvdQ production system (BBa_K855001)

Quorum Sensing Disruption

through pvdQ Production

Overview

1. Introduction 2. Project Design 3. Results 4. Human Practice

Input

Chemical

Signals

Processing Output

Chemical

Signals

MICROBIAL INTELLIGENCE

ANTIBIOTICS

HUMAN INTELLIGENCE

A GLOBAL

CRISIS:

ANTIBIOTIC

RESISTANCE

… but bacteria were more intelligent

A SINGLE

BACTERIUM

A COLONY OF BACTERIA

BIOFILMS

1. Attach to the Surface

2. Secrete Extra Polymeric Substance

3. Signal the Synthesis of Effector Molecules

4. Regulate Pathways -> Hyper Inflammation

BIOFILM FORMATION MECHANISM

http://botserv1.uzh.ch/microbio/site/research/introduction/Biofilm.php

EFFECTS OF BIOFILMS ON BACTERIA

• Slow penetration of toxic substances

• Accelerated horizontal gene transfer

• Slow growth rate

• Metabolically inert state

Antibiotic

Treatment

Not Effective

QUORUM SENSING MECHANISM

1. Reach a threshold cell density

2. AHL production

3. AHL concentration reaches threshold

4. AHL binds to regulatory protein

5. Transcription activation

http://www.lifesci.dundee.ac.uk/groups/sarah_coulthurst/research-areas

Solution

Signal molecule does not reach critical concentration

Virulence protein is not transcribed

AH

L C

on

cen

trat

ion

->

Threshold

LACTONASE vs ACYLASE

Lactonase: Ring opening of AHL -> AHL reforms [under acidic conditions] Acylase: 1. Fatty Acyl Chain: subsequently metabolized 2. Homoserine Lactone: growth inhibitor of bacteria

Pseudomonas. Volume 5: A Model System in Biology, Chapter 15 (2007)

pvdQ Isolation

Pseudomonas

aeruginosa

pvdQ Acylase

http://www.esrf.eu/UsersAndScience/Publications/Highlights/2010/sb/sb19

1. Constitutive pvdQ expression

EXPRESSION SYSTEMS

2. AHL inducible pvdQ expression (BBa_K855001)

EXPRESSION SYSTEMS

lacI+pL luxR pvdQ Lux pR

LuxR pvdQ

AHL

1. Constitutive pvdQ expression

2. AHL inducible pvdQ expression (BBa_K855001)

Cloning pvdQ Expression pvdQ Activity

Assay

Project Plan


Assay

• A 18 bp his tag is attached to the C – terminal of the beta chain

Alpha Beta Signal peptide Spacer peptide

PAO1 pvdQ gene

His

tag

•A 18bp his tag is attached to the C – terminal of the beta chain • Remove the internal PstI site at (1,489-1,494 bp) by silent mutation.

• K855005: CTGCAG > CTCCAG Leucine codon • K855006: CTGCAG > CTGCAA glycin codon

Alpha Beta

Signal peptide Spacer peptide

PAO1 pvdQ gene

His

tag PstI


Assay

1. BBa_ K855000:

2. BBa_ K855001:

3. BBa_ K855002:

pvdQ

pvdQ


Assay

In PROGRESS: 1. BBa_K855005: PstI site removed version of K855000 by site

directed mutagenesis at leucine codon

2. BBa_K855006: PstI site removed version of K855000 by site directed mutagenesis at glycine condon.

3. Bba_K855007: Structural intermediate of AHL inducible system

4. BBa_K855008: AHL inducible production of pvdQ (BBa_K855005)

5. Bba_K855009: AHL inducible production of pvdQ (Bba_K855006)


Assay

BL21 BL21


Assay

Whole Cell Sonication Osmotic Shock


Assay

1. Whole Cell Sonication Osmotic Shock

2. Supernatant

3. Pellet

4. Osmotic Shock Fluid


Assay

1. Whole Cell Sonication Osmotic Shock

2. Supernatant

3. Pellet

4. Osmotic Shock Fluid

SDS-Page

Western Blot using Anti-His tag


Assay

Alpha Beta

Signal peptide Spacer peptide

PAO1 pvdQ gene

His

tag

18 kDa 60 kDa


Assay


Assay

Sonication Whole Cell

Sonication 1. Whole Cell

2. Supernatant

3. Pellet


Assay

Sonication 1. Whole Cell

2. Supernatant

3. Pellet

Controls: -Boiled Fractions :

4. Whole Cell

5. Sonication Supernant

6. Sonication Pellet

7. PBS

-Indication of natural

degradation of AHL


Assay

15 ug AHL 0 ug AHL

100 μl sample

30 ℃ , 4 hours, 70 r.p.m shaker

AHL Assay


Assay

Colorimetric

Determination of intact

AHL concentration

• Quick & Easy • Reliable linearity between the Absorbance (520nm) and AHL concentration

y = 0.0035x + 0.0022 R² = 0.9761

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 50 100 150 200 250 300

Ab

sorb

ance

(5

20

nm

)

Amount of C12 – AHL (ug)

C12-AHL Assay Standard Curve


Assay

• Expressed pvdQ protein is functional. • It displays acylase activity that degrades C12-AHL


Assay

Future Directions 1. Characterize the AHL inducible pvdQ production

system (BBa_K855001) . Construct the constitutive design. Compare the efficiency.

2. Extracellular secretion system of pvdQ.

3. Encapsulation of PseudoKill using alginate beads.

H U M A N

BREAKTHROUGH

OUT OF THE BOX

HAVE SOME FUN!

MAGNIFY… Our audience

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Current

workings

Future

implications

Applications

bio

me

dic

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ag

ricu

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erg

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co

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Video clip Campus TV

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Playing over and

over for 2 weeks!

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SMART MOB!

Distinctive Characters

Dr. Ventor Synthia Moralist

SAY HI TO SYNTHIA !

A HEATED DEBATE…

Dr. Ventor Moralist

Interactions

LEAFLETS

Faculty of Science, Faculty of Medicine,

Faculty of Engineering, Department of Biochemistry

Platinum Sponsor: Gold Sponsor:

Our fellow teammates, advisors and instructors

References:

Bokhove, M., Jimenez, P. N., Quax, W. J., Quax, W. J., & Dijkstra, B. W. (2010). The quorum-quenching n-acyl homoserine lactone acylase pvdq is an ntn-hydrolase with an unusual substrate-binding pocket. PNAS, 107(2), 686–691. Yang, Y. H., Lee, T. H., Kim, J. H., Kim, E. J., Joo, H. S., Lee, C. S., & Kim, B. G. (2006). High-throughput detection method of quorum-sensing molecules by colorimetry and its applications. Analytical Biochemistry, (356), 297-299. Kievit, T. R. (2009). Quorum sensing in pseudomonas aeruginosa biofilms. Environmental Microbiology, 11(2), 279–288. doi: 10.1111/j.1462-2920.2008.01792.x Choi, J. H., & Lee, S. Y. (2004). Secretory and extracellular production of recombinant proteins using escherichia coli. Appl Microbiol Biotechnol, (64), 625–635. doi: 10.1007/s00253-004-1559-9 Wahjudi, M., Mariana, E., Hendrawati, O., Assen, A. H. G. V., Merkerk, R. V., Cool, R. H., & Poelarends, G. J. (2011). Pa0305 of pseudomonas aeruginosa is a quorum quenching acylhomoserine lactone acylase belonging to the ntn hydrolase superfamily.Microbiology, (157), 2042-2055. doi: 10.1099/mic.0.043935-0

Documents

Quorum Sensing Disruption through pvdQ Production2012.igem.org/files/presentation/HKU_HongKong.pdf · 2012-10-06 · 1. Characterize the AHL inducible pvdQ production system (BBa_K855001)