A SynBio Approach to Translational Medicine ¡ ¡
SynBio Approach to Translational Medicine ¡ ¡ Practices ¡ • Useful Bedside ¡ • Responsible Bench ● New approach Clinic
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. ¡ ¡
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene Healthy ¡Airway ¡ Mutant CFTR airway
Importance of Tracking Microbiology Cycles of exacerbation result in accumulating loss of lung function over time 100% Exacerbations are due to an overt immune response to infection Lung function (FEV 1 ) 30% Full Oxygen Support/Lung Transplant List Time (years)
Burden of Care • Physiotherapy • Antibiotics • Digestive enzymes • DNAse to break down mucus • Nebulised medication Isolation and Effect on Mental Wellbeing ¡
Rapid Decline in Health Without Early Intervention “Cough all the time” “Four friends with CF have passed away in the last year” “It’s just lonely” “You can get ill really suddenly”
Current Clinical Need Practices “ 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 only the rear view mirror . ” - Oli Rayner, Special Adviser, Research & Patient Involvement at Cystic Fibrosis Trust
Current Diagnostic Methods Existing methods take up to two weeks ¡ ¡ ¡ ¡ ¡ Quicker Method needed to Preserve Lung ¡ Function ¡ ¡
So Who Are These Bacterial Bandits?
Clinically Critical Burkholderia cenocepacia • Often misdiagnosed • Highly contagious • Can stop people from getting a lung transplant in the UK
The Lung Ranger Existing detection methods take too long Develop a rapid and quantitative method to detect first isolates of Burkholderia cenocepacia.
Our Idea: A Biological Eavesdropper The Lung Ranger will listen in on Burkholderia cenocepacia signals ¡ Burkholderia cenocepacia biofilm ¡ ¡ Time B. cenocepacia B. cenocepacia BDSF
The Lung Ranger The Lung Ranger eavesdrops on bacterial communication signals - a biological detector using synthetic biology. BDSF Sputum
Designing a Biological System to Eavesdrop on BDSF BDSF BCAM0227 [3,036 bp synthetic gene] BCAM0228 Bba_K1315007 ¡ GFP, P cblD , P tat , BCAM0227, BCAM0228 P ¡ ¡ ¡ P cblD RBS GFP BBa_K562012 BBa_K1315008 iGEM Dundee 2011
Characterising the Burkholderia Eavesdropper The biosensor components are produced in an E. coli chassis BCAM0227 HA BCAM0228 HA P ¡ ¡ ¡ P cblD GFP
Characterising the Burkholderia Eavesdropper Problem: GFP produced even without addition of exogenous BDSF BCAM0227 HA BCAM0228 HA P ¡ ¡ ¡ P cblD GFP
Characterising the Burkholderia Eavesdropper Problem: GFP produced even without the BCAM0227 sensor kinase BCAM0228 HA ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ P cblD GFP
Characterising the Burkholderia Eavesdropper Problem: Potential “crosstalk” ? ¡ ? ¡ ? ¡ from chassis kinases or phospho-donors ATP ADP BCAM0228 HA ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ P ¡ Acetyl-P Carbamoyl-P ¡ ¡ P cblD GFP
Clinically Critical Pseudomonas aeruginosa • Occurs in 80% of CF patients by 18 years of age • Severely worsens prognosis • Biofilm highly antibiotic resistant
Our Idea: A Biological Eavesdropper The Lung Ranger will listen in on Pseudomonas aeruginosa signals ¡ Pseudomonas aeruginosa biofilm ¡ ¡ Time P. PQS aeruginosa P. aeruginosa PAI-1 ¡ ¡
Designing a Biological System to Eavesdrop on PQS PQS ¡ PqsR Bba_K1157001 NTU-Taida 2013, Taiwan mCherry, P pqsA P tet PqsR ¡ ¡ P pqsABCDE RBS ¡ mCherry ¡ BBa_K562011 BBa_K1315001 iGEM Dundee 2011
Characterizing the PQS Biosensor The PqsR biosensor is produced in an E. coli chassis pqsR PqsR HA ¡ P pqsABCDE RBS mCherry
Characterizing the PQS Biosensor Problem: Biosensor apparently not responding to PQS pqsR PqsR HA ¡ P pqsABCDE RBS mCherry
Population Level Expression mCherry expression demonstrates sigmoidal response to PQS signal
Most Cells Express No mCherry Are promoters the rate limiting step?
Promoters Limit Expression
Another Method to Eavesdrop on Pseudomonas aeruginosa A biological system that will listen in on Pseudomonas aeruginosa signals ¡ Pseudomonas aeruginosa biofilm ¡ ¡ Time P. PQS aeruginosa P. aeruginosa PAI-1 ¡ ¡
Designing a Biological System to Eavesdrop on PAI-1 Sets of pre-existing BioBricks from Antiquity Boston University used to build two circuits P tet , LasR CDS; P lasB , GFP P tet , LasR CDS; P luxR , GFP LasR LasR Ptet, ¡LasR ¡CDS,P lasR ¡ Ptet, ¡LasR ¡CDS,P lasR ¡ BBa_C0179 BBa_C0179 ¡ ¡ P luxR P lasB GFP GFP BBa_E0040 BBa_R0079 BBa_R0062 BBa_E0040 PAI-1 ¡ ¡
Characterizing the PAI-1 Biosensor LasR LasR BBa_C0179 BBa_C0179 P luxR P lasB GFP GFP BBa_R0062 BBa_R0079 BBa_E0040 BBa_E0040 P lasB -GFP P luxR- GFP Time after PAI-1 0 10 30 60 60 Time after PAI-1 kDa kDa 0 10 30 60 60 addition addition 35 35 GFP GFP 25 25 P lasB /LasR system: P luxR /LasR system: slow induction of GFP over time initially ON, then increased induction of GFP over time
Characterizing the PAI-1 Biosensor LasR LasR BBa_C0179 BBa_C0179 P luxR P lasB GFP GFP BBa_R0062 BBa_R0079 BBa_E0040 BBa_E0040 A Functional Pseudomonas aeruginosa Biosensor!
Local CF Community Practices Clinical Psychologists, Chest Physicians, Physiotherapists, and Nurses CF Clinical Nurse Specialist Lawrie MacDougall Chest Physician Dr James Chalmers CF Clinical Nurse Specialist Gill Brady
National Impact Practices
Concerns Raised ¡ Practices ¡ ¡ 1. How can we implement this detector? 2. How can we do this without releasing GMMs? ¡ ¡ ¡
Making the Lung Ranger a Reality Capturing light from E. coli chassi with an electronic detector . Something which will decrease burden of care
Every Good Ranger Needs a L.A.S.S.O .
Every Good Ranger Needs a L.A.S.S.O .
L ight A mplifying S ignal S ensing O bject A photodetector potentially capable of measuring bacterial load in sputum samples by quantifying the light emitted by our engineered E. coli
The Design Pipeline “From Bedside to Bench and Back Again” Spoke ¡to ¡ Spoke to members ¡of ¡ clinicians and medical ¡industry ¡ CF patients
The Design Pipeline “From Bedside to Bench and Back Again” Spoke to Spoke to members of clinicians and medical CF patients industry
The Design Pipeline “From Bedside to Bench and Back Again” Spoke to Follow ISO for Spoke to members of quality clinicians and medical management CF patients industry
The Design Pipeline “From Bedside to Bench and Back Again” Spoke to Spoke to Follow ISO for Spoke to members of members of Created the quality clinicians and medical medical L.A.S.S.O. management CF patients industry industry
The Design Pipeline “From Bedside to Bench and Back Again” Spoke to members of medical industry
The Design Pipeline “From Bedside to Bench and Back Again” Spoke to members of medical industry
Patient Centred Device Prac)cality ¡ Safety ¡ Waste ¡compartment ¡ Sputum ¡sample ¡ Electronics ¡
The L.A.S.S.O. Interface “the biggest challenge right now is electronic record keeping in hospitals” National Director for Patients and Information in NHS
Socio-economic Impact The Lung Ranger Current 2 hrs 2 weeks Detection Time $24M $12M Hospitalization Cost Recovered GDP Contribution $0 $21M 40 days 20 days Adults: Days Off Work 30 days 15 days Children: Days Off School *Based on CF Registry 2013 Annual Report
Presented to our Local CF Community Practices Clinical Psychologists, Chest Physicians, Physiotherapists, and Nurses CF Clinical Nurse Specialist Lawrie MacDougall Chest Physician Dr James Chalmers CF Clinical Nurse Specialist Gill Brady
The Lung Ranger Science Summary • Basic research into bacterial communication • Functioning PAI-1 biosensor • Electronic device and L.A.S.S.O. Interface Future Work • Detect signals from clinical samples (sputum) • Sense more pathogens in CF • Implement L.A.S.S.O. into other diseases ¡
SynBio approach to translational medicine ¡ Practices ¡ • Useful -Addressing an unmet need identified by patients, professionals and Governing bodies (CF Trust). • Responsible -Bedside to Bench and Back again. • New approach -Developed a blueprint for responsible use of SynBio to meet societal needs.
¡ School ¡of ¡CompuAng ¡ School ¡of ¡EPM ¡ School ¡of ¡Life ¡Sciences ¡ ¡ DNA ¡Sequencing ¡ Services ¡ ¡ ¡ ¡ ¡THANK ¡YOU ¡ ¡
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