Abstract Topoisomerase Production Pipeline2012.igem.org/files/poster/Arizona_State.pdf · This gel...
1
Mass spectrometry measurements of our His-purified BL21 Control Lysate and Topoisomerase D168A protein sample are shown below. Peaks at 2428, 1750 and 2889 show a strong similarity between the topoisomerase protein sample and the expect peptide fragments based on the topoisomerase D168A amino acid sequence. This measurement confirms that our protein isolate does in fact contain the topoisomerase mutant protein. Undergraduates: Hyder Hussain, Amanda Ispas, Abhinav Markus, Ryan Muller, Nisarg Patel, Ellen Qin, Rohit Rajan, Madeline Sands, Ethan Ward. Advisors: Karmella Haynes, Xiao Wang, Vincent Pizziconi. Diarrheic pathogens including E. coli O157:H7 serotype, Campylobacter, Shigella, and Salmonella often contaminate drinking water supplies in developing nations and are responsible for approximately 1.5 million worldwide annual deaths. Current technologies for detection of bacteria include DNA hybridization FRET signaling, electrical detection via immobilized antimicrobial peptides, and PCR amplification followed by gel visualization. Our method of bacterial detection fills a niche in biosensor technology. Our design implies lower costs, higher portability, and a more rapid signal output than most bacterial biosensors. Additionally, our interchangeable DNA probe confers modularity, allowing for a range of bacterial detection. Using a novel split beta-galactosidase complementation assay, we have designed three unique chimeric proteins that recognize and bind to specific pathogenic markers and create a functioning beta-galactosidase enzyme. This functioning enzyme unit then cleaves X-gal and produces a colorimetric output signal. Our research demonstrates success in initial stages of chimeric protein assembly. Abstract Topoisomerase From Start to Finish: Production Pipeline Viewed as a minor inconvenience in the developed world, diarrhea can be a death sentence in developing countries. Diarrhea can be life threatening as it causes severe dehydration as a result of extensive fluid loss. An estimated 2.0 billion cases of diarrhea occur each year amongst children under five years of age. Of these cases, 1.5 million children die. Motivation Our biosensor design module consist of two split fragments of the enzyme beta-galactosidase: alpha and omega. A sensor of choice—whether DNA or cell surface targeting—can be fused to either fragment of b-gal. If no pathogen is present in a sample, the sensors will not bind anything and the complete, functional b-gal unit will not be formed. In the presence of the target pathogen, multiple sensors will bind to the target, bringing together the split enzyme fragments attached to the sensors. Results confirmed that this will create a functional b-gal unit. In the presence of an added substrate (x-gal), a fully functional enzyme unit will cleave the substrate and turn the sample blue, indicating that the sample is contaminated. This design can be adapted to detect a variety of pathogens by simply swapping the sensor component. Modularity Target Sequences Achievements Acknowledgments Determining whether or not water is safe to drink is an essential step in minimizing disease cases. Clean water is not always readily available and natural disasters often contaminate water sources without notice. The ASU iGEM team is developing an inexpensive way for communities to test the purity and identify the specific pathogens in water sources to reduce the incidence of childhood diarrhea and ultimately decrease mortality rates. Specifically, our design strategy involved developing biosensors to detect both DNA and cell surface antigens. Biosensor design modules with the appropriate ligand are expressed in E. coli, which acts as a biosensor production factory. The sensors are then purified and added to a heat-treated water sample to hybridize with target sequences and produce a response. Human Practices Mathematical Modeling: Used Bayes’ Rule to estimate sensitivity and specificity of a biosensor as well as the and natural frequency of a disease outbreak. Bayes’ rule gives us the probability of an actual positive event given that our sensor output is "true". Ethical Considerations: Discussed the ethical dilemma of having the knowledge of contaminated supply sources but being unable to treat the water itself following detection. Escherichia Coli Case Studies: Researched and described how our final biosensor would be utilized in real world scenarios compared to current practices and its community impact. International Outreach: Developed and implemented a water hygiene program in Guatemala. Community Outreach: Mentored summer research interns and assisted Bioscience High School with their iGEM project in the high school competition. Built, characterized, and submitted 3 Biobricks that worked as expected Because our team works in a BSL1 lab, we are unable to test our biosensors on live pathogenic bacteria. To demonstrate proof-of-concept, we identified an E. coli strain in the Keio family that has GFP expression integrated in its genome. We then identified two 20 base pair sequences spaced 10 base pairs apart in this sequence that were unique to these sites in the strain's genome. Our Biobricks BBa_K891000 and BBa_K891999 each contain two repeats of one of the two 20 base pair recognition sequences flanked on either side by the CCCTT topoisomerase binding sites. By co-transforming these Biobricks along with BBa_K891234, which codes for the mutant form of topoisomerase, we plan to generate our entire bgal-protein-ssDNA probe generated in vivo for easy isolation using a nickel binding column via His-purification. We can then treat water samples containing the Keio strain with these probes and characterize the expected colorimetric response associated with the ssDNA hybridization with the target's genomic DNA. Confirmed feasibility of split beta-galactosidase as a reporter system. Developed a model for biosensor feasibility based upon past outbreaks and created a hygiene program for preventative care post-detection. • Dr. Karmella Haynes • Dr. Vincent Pizziconi • Dr. Xiao Wang • Dr. Miles Orchinik • Dr. Marco Santello • Dr. Thomas E. Grys • Kylie Standage-Beier • Alizee Jenck • Jordan Nguyen • James Alling • Rene Davis • Eric Trang • Arizona State University • Case Western Reserve University • University of Pennsylvania • Deans Paul Johnson, Jim Collofello • Dean Mark Jacobs • ASU President Michael Crow Xgal Sensor alpha omega We used topoisomerase to nick a specific covalently bonded sequence and peel off a section of ssDNA. We have designed a template plasmid that includes tandem YCCTT recognition sites with template strand in between, and is complementary to a section of coding sequence of GFP. By inducing topoisomerase/split b-gal fusion protein expression, we can generate chimeric probes in vivo that can be easily His-tag purified and used for detection. His-purified BL21 Control Lysate Topoisomerase D168A We incubated GFPT2 with various protein samples. This gel shows that our protein isolate contains functional protein and that our mutant and wild-type proteins behave as expected. We see plasmid supercoiling in the DNA and control lysate wells (Wells 1 and 2), fully relieved super coiling from the wild-type topoisomerase (Well 3) and highly fragmented DNA from the mutant topoisomerase (Well 4) because it did not re-ligate the DNA after creating nicks, showing that the mutant protein behaved as expected. The last well contains the topoisomerase mutant with no DNA.
Transcript of Abstract Topoisomerase Production Pipeline2012.igem.org/files/poster/Arizona_State.pdf · This gel...
Mass spectrometry measurements of our His-purified BL21 Control Lysate and Topoisomerase D168A protein sample are shown below. Peaks at 2428, 1750 and 2889 show a strong similarity between the topoisomerase protein sample and the expect peptide fragments based on the topoisomerase D168A amino acid sequence. This measurement confirms that our protein isolate does in fact contain the topoisomerase mutant protein.
Undergraduates: Hyder Hussain, Amanda Ispas, Abhinav Markus, Ryan Muller, Nisarg Patel, Ellen Qin, Rohit Rajan, Madeline Sands, Ethan Ward.
Advisors: Karmella Haynes, Xiao Wang, Vincent Pizziconi.
Diarrheic pathogens including E. coli O157:H7 serotype, Campylobacter, Shigella, and Salmonella often contaminate drinking water supplies in developing nations and are responsible for approximately 1.5 million worldwide annual deaths. Current technologies for detection of bacteria include DNA hybridization FRET signaling, electrical detection via immobilized antimicrobial peptides, and PCR amplification followed by gel visualization. Our method of bacterial detection fills a niche in biosensor technology. Our design implies lower costs, higher portability, and a more rapid signal output than most bacterial biosensors. Additionally, our interchangeable DNA probe confers modularity, allowing for a range of bacterial detection. Using a novel split beta-galactosidase complementation assay, we have designed three unique chimeric proteins that recognize and bind to specific pathogenic markers and create a functioning beta-galactosidase enzyme. This functioning enzyme unit then cleaves X-gal and produces a colorimetric output signal. Our research demonstrates success in initial stages of chimeric protein assembly.
Abstract Topoisomerase From Start to Finish:
Production Pipeline
Viewed as a minor inconvenience in the developed world, diarrhea can be a death sentence in developing countries. Diarrhea can be life threatening as it causes severe dehydration as a result of extensive fluid loss. An estimated 2.0 billion cases of diarrhea occur each year amongst children under five years of age. Of these cases, 1.5 million children die.
Motivation
Our biosensor design module consist of two split fragments of the enzyme beta-galactosidase: alpha and omega. A sensor of choice—whether DNA or cell surface targeting—can be fused to either fragment of b-gal. If no pathogen is present in a sample, the sensors will not bind anything and the complete, functional b-gal unit will not be formed. In the presence of the target pathogen, multiple sensors will bind to the target, bringing together the split enzyme fragments attached to the sensors. Results confirmed that this will create a functional b-gal unit. In the presence of an added substrate (x-gal), a fully functional enzyme unit will cleave the substrate and turn the sample blue, indicating that the sample is contaminated. This design can be adapted to detect a variety of pathogens by simply swapping the sensor component.
Modularity Target Sequences
Achievements
Acknowledgments
Determining whether or not water is safe to drink is an essential step in minimizing disease cases. Clean water is not always readily available and natural disasters often contaminate water sources without notice. The ASU iGEM team is developing an inexpensive way for communities to test the purity and identify the specific pathogens in water sources to reduce the incidence of childhood diarrhea and ultimately decrease mortality rates. Specifically, our design strategy involved developing biosensors to detect both DNA and cell surface antigens.
Biosensor design modules with the appropriate ligand are expressed in E. coli, which acts as a biosensor production factory. The sensors are then purified and added to a heat-treated water sample to hybridize with target sequences and produce a response.
Human Practices Mathematical Modeling: Used Bayes’ Rule to estimate sensitivity and specificity of a biosensor as well as the and natural frequency of a disease outbreak. Bayes’ rule gives us the probability of an actual positive event given that our sensor output is "true".
Ethical Considerations: Discussed the ethical dilemma of having the knowledge of contaminated supply sources but being unable to treat the water itself following detection. Escherichia Coli Case Studies: Researched and described how our final biosensor would be utilized in real world scenarios compared to current practices and its community impact. International Outreach: Developed and implemented a water hygiene program in Guatemala. Community Outreach: Mentored summer research interns and assisted Bioscience High School with their iGEM project in the high school competition.
Built, characterized, and submitted 3 Biobricks that worked as expected
Because our team works in a BSL1 lab, we are unable to test our biosensors on live pathogenic bacteria. To demonstrate proof-of-concept, we identified an E. coli strain in the Keio family that has GFP expression integrated in its genome. We then identified two 20 base pair sequences spaced 10 base pairs apart in this sequence that were unique to these sites in the strain's genome. Our Biobricks BBa_K891000 and BBa_K891999 each contain two repeats of one of the two 20 base pair recognition sequences flanked on either side by the CCCTT topoisomerase binding sites.
By co-transforming these Biobricks along with BBa_K891234, which codes for the mutant form of topoisomerase, we plan to generate our entire bgal-protein-ssDNA probe generated in vivo for easy isolation using a nickel binding column via His-purification. We can then treat water samples containing the Keio strain with these probes and characterize the expected colorimetric response associated with the ssDNA hybridization with the target's genomic DNA.
Confirmed feasibility of split beta-galactosidase as a reporter system.
Developed a model for biosensor feasibility based upon past outbreaks and created a hygiene program for preventative care post-detection.
• Dr. Karmella Haynes • Dr. Vincent Pizziconi • Dr. Xiao Wang • Dr. Miles Orchinik • Dr. Marco Santello • Dr. Thomas E. Grys
• Kylie Standage-Beier • Alizee Jenck • Jordan Nguyen • James Alling • Rene Davis • Eric Trang
• Arizona State University • Case Western Reserve University • University of Pennsylvania
• Deans Paul Johnson, Jim Collofello • Dean Mark Jacobs • ASU President Michael Crow
X-‐gal
Sensor
alpha
omega
We used topoisomerase to nick a specific covalently bonded sequence and peel off a section of ssDNA. We have designed a template plasmid that includes tandem YCCTT recognition sites with template strand in between, and is complementary to a section of coding sequence of GFP. By inducing topoisomerase/split b-gal fusion protein expression, we can generate chimeric probes in vivo that can be easily His-tag purified and used for detection.
His-purified BL21 Control Lysate Topoisomerase D168A
We incubated GFPT2 with various protein samples. This gel shows that our protein isolate contains functional protein and that our mutant and wild-type proteins behave as expected. We see plasmid supercoiling in the DNA and control lysate wells (Wells 1 and 2), fully relieved super coiling from the wild-type topoisomerase (Well 3) and highly fragmented DNA from the mutant topoisomerase (Well 4) because it did not re-ligate the DNA after creating nicks, showing that the mutant protein behaved as expected. The last well contains the topoisomerase mutant with no DNA.
