Genetically-Engineered Bacteriophage for Diagnosis of...
1
Results Experimental Workflow Conclusions Human Practices Modifying the Phage Genome Infection Period (Days) Peak of Epidemic Positive controls Stock hCG dimer Negative control No hCG present Positive results Applied directly to test After denaturing and folding, both bands are fainter No band Genetically-Engineered Bacteriophage for Diagnosis of Whooping Cough BioBricks • hCGβ was PCR-amplified from a commercial vector and cloned into the pSB1C3 backbone • The putative lysin bbp9 was PCR-amplified from Bordetella phage genome and then cloned • synCGβ was codon-optimized in silico, chemically synthesized, and cloned. • A strong T7 promoter and RBS were inserted in front of synCGβ Detecting hCGβ • E. coli with pET30a vector containing hCGβ under the control of a strong promoter and lac operator were induced with IPTG. • Denatured & refolded protein was applied, and the control band appeared. Test band was visible the next day. • Test band was not observed when extract was applied without folding solution. A Novel Diagnostic Epidemiological Model Josh Fass 1 , Jacqueline Grimm 2 , Syed Hassan 3 , John Hubczak 4 , Joseph Muldoon 2,5 , Shaun Moshasha 5,6 , Omar Raza 2 and Alexander Zorychta 1 • QUICK AND EASY. One-day diagnostic, coupling optimization of pregnancy tests to sense trace amounts of hCG and the speed of bacteriophage replication inside a host cell. • SPECIFIC. Due to host-pathogen coevolution, bacteriophages have high specificity. • IMPLEMENTABLE IN THE DEVELOPING WORLD. Pregnancy tests are widely available, easy to use, and cost $1. Whooping Cough: A Resurging Threat Acknowledgements & Sponsors • Highly contagious respiratory disease • 16 million annual cases worldwide • 95% of cases in developing world • Major cause of infant mortality – 195,000 per year 1 • Major U.S. epidemics in 2005, 2010, and 2012 2 • Current diagnostic methods are inadequate iGEM Advisors: Drs. Keith Kozminski, Jason Papin, and Inchan Kwon Kelly Laboratory: Dr. Kimberly Kelly, Dr. Siva Dasa, Dr. Jeff Smith, Stephanie Thomas, Lindsey Brinton, Marc Seaman, Soo Shin, Jaymes Beech, and Dustin Bauknight Hewlett Laboratory: Eric Hewlett, MD, Dr. Gina Donato Arjun Athreya, Yanzhi Yang, Jacqueline Niu UVA Alumni Lacey Fund Deans Hawley and Galloway of the UVA College of Arts and Sciences UVA Medical School Progression of epidemic (inflections per time) over a range of infection periods. The arrow tracks the peak of epidemic over different infection periods. The peak becomes lower and delayed when the infection period is shorter. Implications of Model In an epidemic, shorter infection period due to rapid diagnosis: • Decreases total infected individuals. • Delays peak of epidemic (maximum of the infected individuals) to delay to a later time Compartmental Model of Variable Populations S = Susceptible E = Exposed I = Infected R = Recovered T = Treated Collaboration • Offered and solicited skills using NTNU Trondheim’s iGEM Matchmaker • Provided feedback on Team Grenoble’s BioBrick Safety Sheet (BSS) Outreach • Organized a Flash Seminar with member of Presidential Bioethics Commission • Generated local excitement about synbio Intellectual Property Analysis • Compared alternative intellectual property policies for synbio • Advocate development of a sui generis IP framework conceptually adequate to technical reality Metaphor Analysis • Metaphors inevitable and influential in policy, public perception, and design intuition • Cognitive science should inform representational design • Reductionist framing can help overcome representations’ functional fixedness bbp9 CAT BBa_K732003 hCG! CAT BBa_K732000 synCG! CAT BBa_K732001 synCG! CAT BBa_K732002 P RBS hCG! kan pET-30a construct P O lacI • Novel diagnostic for bacterial disease using engineered bacteriophage and pregnancy tests • Demonstrate expression and detection of a codon-optimized hCG-β by $1 pregnancy tests after the application of a protein-folding solution Next: • Completing the T7 phage modification • Optimizing the refolding solution for clinical use • Expanding to the detection of pertussis and other organisms. University of Virginia Departments of Biomedical Engineering 1 , Biology 2 , Computer Science 3 , Chemical Engineering 4 , Chemistry 5 , and Physics 6 Controls • Positive control: when hCG dimer was applied to a pregnancy test, a bright test band resulted. • To detect protein that does not fold properly in the bacterial cytosol, denaturing solution and folding solution were applied to cell lysate • When the dimer was denatured and refolded, the resulting band was duller and took longer to appear • The folding solution lessens the band intensity and lengthens detection time; however, it is necessary because the protein does not fold properly in bacteria • Negative control: no band in the absence of hCG protein Detecting synCGβ Cells containing a heterogeneous mixture of BBa_K732001 and BBa_K732002 were lysed by either T7 infection or sonication, and synCGβ was denatured and refolded. Positive test bands resulted in both cases. Sonication T7 infection T7 Assembly from Genome • Chemically competent E. coli were transformed with T7 genome. • Cells began lysing after several hours, indicating assembly of intact, infectious phage. • Proof of principle: modified phage could be assembled from modified genome using transformation References 1. World Health Organization (2011). Pertussis. Retrieved from http://www.who.int/immunization/topics/pertussis/en/index.html. 2. Cherry, J. D. (2012). Epidemic Pertussis in 2012 - The Resurgence of a Vaccine-Preventable Disease. The New England Journal of Medicine, 267(9), 785–787. • Clinical diagnosis - early symptoms nondescript • Lab detection • PCR - prone to false positives, expensive • Culturing - takes several days • Serology – useful after 2-8 weeks
Transcript of Genetically-Engineered Bacteriophage for Diagnosis of...
Results
Experimental Workflow
Conclusions
Human Practices
Modifying the Phage Genome
Infection Period (Days)!
Peak of Epidemic
Positive controls!Stock hCG dimer!
Negative control!No hCG present!
Positive results!
Applied directly to test! After denaturing and folding,!both bands are fainter !
No band!
Genetically-Engineered Bacteriophage for Diagnosis of Whooping Cough
BioBricks • hCGβ was PCR-amplified from a commercial
vector and cloned into the pSB1C3 backbone • The putative lysin bbp9 was PCR-amplified from
Bordetella phage genome and then cloned • synCGβ was codon-optimized in silico, chemically
synthesized, and cloned. • A strong T7 promoter and RBS were inserted in
front of synCGβ
Detecting hCGβ • E. coli with pET30a vector containing hCGβ under the
control of a strong promoter and lac operator were induced with IPTG.
• Denatured & refolded protein was applied, and the control band appeared. Test band was visible the next day.
• Test band was not observed when extract was applied without folding solution.
A Novel Diagnostic
Epidemiological Model
Josh Fass1, Jacqueline Grimm2, Syed Hassan3, John Hubczak4, Joseph Muldoon2,5, Shaun Moshasha5,6, Omar Raza2 and Alexander Zorychta1
• QUICK AND EASY. One-day diagnostic, coupling optimization of pregnancy tests to sense trace amounts of hCG and the speed of bacteriophage replication inside a host cell.
• SPECIFIC. Due to host-pathogen coevolution, bacteriophages have high specificity.
• IMPLEMENTABLE IN THE DEVELOPING WORLD. Pregnancy tests are widely available, easy to use, and cost $1.
Whooping Cough: A Resurging Threat
Acknowledgements & Sponsors
• Highly contagious respiratory disease • 16 million annual cases worldwide • 95% of cases in developing world • Major cause of infant mortality – 195,000 per year1 • Major U.S. epidemics in 2005, 2010, and 20122
• Current diagnostic methods are inadequate
iGEM Advisors: Drs. Keith Kozminski, Jason Papin, and Inchan Kwon Kelly Laboratory: Dr. Kimberly Kelly, Dr. Siva Dasa, Dr. Jeff Smith, Stephanie Thomas, Lindsey Brinton, Marc Seaman, Soo Shin, Jaymes Beech, and Dustin Bauknight Hewlett Laboratory: Eric Hewlett, MD, Dr. Gina Donato Arjun Athreya, Yanzhi Yang, Jacqueline Niu
UVA Alumni Lacey Fund Deans Hawley and Galloway of the UVA College of Arts and Sciences UVA Medical School
Progression of epidemic (inflections per time) over a range of infection periods. The arrow tracks the peak of epidemic over different infection periods. The peak becomes lower and delayed when the infection period is shorter.
Implications of Model In an epidemic, shorter infection period due to rapid diagnosis: • Decreases total infected individuals. • Delays peak of epidemic (maximum of the infected individuals) to delay to a later time
Compartmental Model of Variable Populations S = Susceptible E = Exposed I = Infected R = Recovered T = Treated
Collaboration • Offered and solicited skills using NTNU Trondheim’s iGEM Matchmaker • Provided feedback on Team Grenoble’s BioBrick Safety Sheet (BSS) Outreach • Organized a Flash Seminar with member of Presidential Bioethics Commission • Generated local excitement about synbio Intellectual Property Analysis • Compared alternative intellectual property policies for synbio • Advocate development of a sui generis IP framework conceptually adequate to technical reality Metaphor Analysis • Metaphors inevitable and influential in policy, public perception, and design intuition • Cognitive science should inform representational design • Reductionist framing can help overcome representations’ functional fixedness
!
bbp9
CAT
BBa_K732003
hCG!
CAT
BBa_K732000
synCG!
CAT
BBa_K732001
synCG!
CAT
BBa_K732002
P RBS
hCG!
kan
pET-30a construct
P O
lacI
hCG! synCG!
!
bbp9
CAT
BBa_K732003
hCG!
CAT
BBa_K732000
synCG!
CAT
BBa_K732001
synCG!
CAT
BBa_K732002
P RBS
hCG!
kan
pET-30a construct
P O
lacI
hCG! synCG!
• Novel diagnostic for bacterial disease using engineered bacteriophage and pregnancy tests
• Demonstrate expression and detection of a codon-optimized hCG-β by $1 pregnancy tests after the application of a protein-folding solution
Next: • Completing the T7 phage modification • Optimizing the refolding solution for clinical use • Expanding to the detection of pertussis and other organisms.
University of Virginia Departments of Biomedical Engineering1, Biology2, Computer Science3, Chemical Engineering4, Chemistry5, and Physics6
Controls • Positive control: when hCG dimer was applied to a
pregnancy test, a bright test band resulted. • To detect protein that does not fold properly in the
bacterial cytosol, denaturing solution and folding solution were applied to cell lysate
• When the dimer was denatured and refolded, the resulting band was duller and took longer to appear
• The folding solution lessens the band intensity and lengthens detection time; however, it is necessary because the protein does not fold properly in bacteria
• Negative control: no band in the absence of hCG protein
Detecting synCGβ Cells containing a heterogeneous mixture of BBa_K732001 and BBa_K732002 were lysed by either T7 infection or sonication, and synCGβ was denatured and refolded. Positive test bands resulted in both cases.
Sonication T7 infection
T7 Assembly from Genome • Chemically competent E. coli were transformed with T7 genome. • Cells began lysing after several hours, indicating assembly of intact,
infectious phage. • Proof of principle: modified phage could be assembled from
modified genome using transformation
References 1. World Health Organization (2011). Pertussis. Retrieved from http://www.who.int/immunization/topics/pertussis/en/index.html. 2. Cherry, J. D. (2012). Epidemic Pertussis in 2012 - The Resurgence of a Vaccine-Preventable Disease. The New England Journal of Medicine, 267(9), 785–787.
• Clinical diagnosis - early symptoms nondescript • Lab detection
• PCR - prone to false positives, expensive • Culturing - takes several days • Serology – useful after 2-8 weeks
![Bacteriophage [Compatibility Mode] (2)](https://static.fdocuments.in/doc/165x107/577cd7461a28ab9e789e8922/bacteriophage-compatibility-mode-2.jpg)
![BACTERIOPHAGE-RESISTANT AND BACTERIOPHAGE-SENSITIVE ...halsmith/phagemutantsubmitted_2.pdf · BACTERIOPHAGE-RESISTANT AND BACTERIOPHAGE-SENSITIVE BACTERIA IN A CHEMOSTAT ... [22],](https://static.fdocuments.in/doc/165x107/5b3839687f8b9a5a518d2ce1/bacteriophage-resistant-and-bacteriophage-sensitive-halsmithphagemutantsubmitted2pdf.jpg)
