[PPT] - A SynBio Approach to Translational Medicine SynBio Approach to PowerPoint Presentation

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A SynBio Approach to Translational Medicine ¡

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Useful

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Practices

SynBio Approach to Translational Medicine ¡

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Bedside Bench Clinic

Responsible
New approach

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Cystic Fibrosis

Common Autosomal

Recessive Disorder

1 in 25 in the UK are

carriers

50% of people with CF

will die before they are 40 years old.

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Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene

Mutant CFTR airway

Healthy ¡Airway ¡

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Importance of Tracking Microbiology

Lung function (FEV1) Time (years) Exacerbations are due to an overt immune response to infection

Cycles of exacerbation result in accumulating loss of lung function

ver time

Full Oxygen Support/Lung Transplant List

30% 100%

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Physiotherapy
Antibiotics
Digestive enzymes
DNAse to break down mucus
Nebulised medication

Isolation and Effect on Mental Wellbeing

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Burden of Care

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“Cough all the time” “It’s just lonely” “Four friends with CF have passed away in the last year” “You can get ill really suddenly”

Rapid Decline in Health Without Early Intervention

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Practices

Current Clinical Need

“ Would be really clinically useful to have a device to detect the first isolates of Burkholderia cenocepacia “

– Dr James Chalmers

Current methods of detection are “like driving a car using

nly the rear view mirror.”
Oli Rayner, Special Adviser, Research &

Patient Involvement at Cystic Fibrosis Trust

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Current Diagnostic Methods

Existing methods take up to two weeks

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Quicker Method needed to Preserve Lung Function

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So Who Are These Bacterial Bandits?

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Clinically Critical

Burkholderia cenocepacia

Often misdiagnosed
Highly contagious
Can stop people from getting a lung

transplant in the UK

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The Lung Ranger

Develop a rapid and quantitative method to detect first isolates of Burkholderia cenocepacia. Existing detection methods take too long

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The Lung Ranger will listen in on Burkholderia cenocepacia signals

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B. cenocepacia Burkholderia cenocepacia biofilm B. cenocepacia BDSF

Our Idea: A Biological Eavesdropper

Time

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The Lung Ranger

The Lung Ranger eavesdrops on bacterial communication signals

a biological detector using synthetic biology.

BDSF Sputum

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Designing a Biological System to Eavesdrop on BDSF

P ¡

BCAM0227

[3,036 bp synthetic gene]

BCAM0228

Bba_K1315007

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¡ ¡PcblD RBS GFP

BBa_K1315008 BBa_K562012

iGEM Dundee 2011

GFP, PcblD, Ptat, BCAM0227, BCAM0228

BDSF

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P ¡

BCAM0227HA BCAM0228HA

¡ ¡PcblD GFP The biosensor components are produced in an E. coli chassis

Characterising the Burkholderia Eavesdropper

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Problem: GFP produced even without addition of exogenous BDSF

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BCAM0227HA BCAM0228HA

¡ ¡PcblD GFP

Characterising the Burkholderia Eavesdropper

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Problem: GFP produced even without the BCAM0227 sensor kinase

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BCAM0228HA

¡ ¡PcblD GFP

Characterising the Burkholderia Eavesdropper

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Problem: Potential “crosstalk” from chassis kinases or phospho-donors

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Acetyl-P Carbamoyl-P ATP ADP GFP

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¡ ¡PcblD

BCAM0228HA

P ¡

Characterising the Burkholderia Eavesdropper

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Clinically Critical

Pseudomonas aeruginosa

Occurs in 80% of CF patients by

18 years of age

Severely worsens prognosis
Biofilm highly antibiotic resistant

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Our Idea: A Biological Eavesdropper

The Lung Ranger will listen in on Pseudomonas aeruginosa signals

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P. aeruginosa P. aeruginosa

Pseudomonas aeruginosa biofilm PQS PAI-1 ¡ ¡ Time

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Designing a Biological System to Eavesdrop on PQS

PQS ¡

PqsR

Bba_K1157001 NTU-Taida 2013, Taiwan

mCherry, PpqsA Ptet PqsR

¡ ¡PpqsABCDE RBS ¡ mCherry ¡

BBa_K1315001 BBa_K562011

iGEM Dundee 2011

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The PqsR biosensor is produced in an E. coli chassis PqsRHA pqsR

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RBS mCherry PpqsABCDE

Characterizing the PQS Biosensor

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PqsRHA pqsR

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Problem: Biosensor apparently not responding to PQS RBS mCherry PpqsABCDE

Characterizing the PQS Biosensor

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Population Level Expression

mCherry expression demonstrates sigmoidal response to PQS signal

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Most Cells Express No mCherry

Are promoters the rate limiting step?

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Promoters Limit Expression

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A biological system that will listen in on Pseudomonas aeruginosa signals

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PQS PAI-1 ¡ ¡

Another Method to Eavesdrop on Pseudomonas aeruginosa

P. aeruginosa P. aeruginosa

Pseudomonas aeruginosa biofilm Time

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Designing a Biological System to Eavesdrop on PAI-1

Ptet, ¡LasR ¡CDS,PlasR ¡

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LasR

BBa_C0179 Ptet, LasR CDS; PluxR, GFP BBa_R0062 BBa_E0040

PluxR GFP

Ptet, ¡LasR ¡CDS,PlasR ¡

¡

LasR

BBa_C0179 BBa_E0040

GFP PlasB

BBa_R0079 Ptet, LasR CDS; PlasB, GFP

Sets of pre-existing BioBricks from Antiquity Boston University used to build two circuits PAI-1 ¡ ¡

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Characterizing the PAI-1 Biosensor

PlasB/LasR system:

slow induction of GFP over time

25 35 kDa GFP 0 10 30 60 60 PlasB-GFP Time after PAI-1 addition 25 35 kDa GFP 0 10 30 60 60 PluxR-GFP Time after PAI-1 addition

PluxR/LasR system:

initially ON, then increased induction of GFP over time

LasR

BBa_C0179 BBa_E0040

GFP PlasB

BBa_R0079

LasR

BBa_C0179 BBa_E0040

GFP PluxR

BBa_R0062

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A Functional Pseudomonas aeruginosa Biosensor!

LasR

BBa_C0179 BBa_E0040

GFP PlasB

BBa_R0079

LasR

BBa_C0179 BBa_E0040

GFP PluxR

BBa_R0062

Characterizing the PAI-1 Biosensor

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Practices

Local CF Community

Chest Physician Dr James Chalmers CF Clinical Nurse Specialist Lawrie MacDougall CF Clinical Nurse Specialist Gill Brady Clinical Psychologists, Chest Physicians, Physiotherapists, and Nurses

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Practices

National Impact

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Practices

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1. How can we implement this detector?
2. How can we do this without releasing

GMMs? ¡ ¡ ¡

Concerns Raised

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Capturing light from E. coli chassi with an electronic detector. Something which will decrease burden of care

Making the Lung Ranger a Reality

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Every Good Ranger Needs a L.A.S.S.O.

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Every Good Ranger Needs a L.A.S.S.O.

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Light Amplifying Signal Sensing Object

A photodetector potentially capable of measuring bacterial load in sputum samples by quantifying the light emitted by our engineered E. coli

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