myn.u. p.a.l ...2011.igem.org/files/poster/northwestern.pdf · title: microsoft powerpoint - truly...
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MY N.U. P.A.L.NORTHWESTERN UNIVERSITY PSEUDOMONAS AERUGINOSA LOCATOR________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
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Valerie Chen, Nirmit Desai, Rafay Faruqi, Kristin Palarz, Helen Shen, Michael ShererAdvisers: Dr. Michael Jewett, Dr. Joshua Leonard, Dr. John Mordacq, Dr. Keith Tyo
Biosensor constructs exhibited significant fluorescence in the presence ofautoinducer molecules
Steady state fluorescence was reached in approximately two hours Characterization of existing and newly isolated promoters identified excellent
candidates for both binary and concentration dependent testing
Testing Methods
System Overview
Mathematical Model
Conclusions
Goal: design biosensor capable of detecting Pseudomonas Aeruginosa quickly,inexpensively, and effectively Pseudomonas Aeruginosa is known to cause a wide variety of severe nosocomial
infections, especially in immunocompromised patients We achieved our goal by engineering a biosensor system in E. coli, using
components from the P. Aeruginosa quorum sensing system Our biosensor constructs detect and report the presence of autoinducer molecules
unique to the P. Aeruginosa quorum sensing system Our biosensor constructs reached steady state in approximately two hours, which
is much faster than other existing detection methods
Summary
Biobrick Assemblies: Las/PAI‐1 constructs provide an excellent binary test for
presence of P. Aeruginosa autoinducers RhlR/PAI‐2 constructs yield output fluorescence readings
proportional to autoinducer concentrationNovel Parts:• Newly isolated genomic promoters provide novel
mechanisms for detecting P. Aerguinosa
We created a video discussing economic and ethical implications of using syntheticbiology in a clinical setting. The video features conversations with: Dr. Dale Mortensen, Nobel Laureate in Economics Dr. Keith Tyo, Synthetic Biologist Dr. Laurie Zoloth, BioethicistWe showed our video in the Northwestern University Technological Institute lobby toget students thinking about synthetic biology.
Human Practices
Applications
Our system’s success meansMy N.U. P.A.L.will have many advantages: Easily portable No expensive or complicated equipment Fast (1‐2 hr) detection of Pseudomonas AeruginosaThedevicecouldbeusedinseveralwaystoreducetheimpactofinfections: Checking hospital equipment for contamination Determining identity of blood or tissue sample Checking hospital rooms before a patient enters
References
Results
Schematicoverviewofphotodiode‐basedclinicaldetector
1. ConstructsconstitutivelyexpressLasorRhl receptorproteins
2. Whenappropriateautoinducerispresent,autoinducerdimerizeswithreceptorprotein
Basalfluorescenceinsignificant,easilydiscernablefrominducedsamples Wellsuitedforbinarytest
PAI‐1 Biosensor: Binary Detection System
PAI‐2 Biosensor: Concentration Sensitive Detection System
Distinguishablefromnegativecontrolsandeachother Discriminatesbetweendifferentautoinducerconcentrations
PAI‐2 Biosensor 2: Newly Isolated Genomic Promoter Construct (Novel Entry)
Superiordiscriminationbetweendifferentautoinducerconcentrations Discriminatesbetweeninitialandsteadystateautoinducerconcentrations
1) Van Delden C, Iglewski BH. Cell‐to‐Cell Signaling and Pseudomonas aeruginosa Infections. Emerging Infectious Diseases. 1998;Vol.4,No.4:551‐560.2) Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, et al. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature.2000;406:959–964.3) Passador L, Cook JM, Gambello MJ, Rust L, Iglewski BH. Expression of Pseudomonas aeruginosa virulence genes requires cell‐to‐cell communication. Science 1993;260:1127‐30.4) Pesci EC, Pearson JP, Seed PC, Iglewski BH. Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa. J Bacteriol 1997;179:3127‐32.•The study resulting in this presentation was assisted by a grant from the Undergraduate Engagement Grant Program which is administered by Northwestern University's Office ofthe Provost. However, the conclusions, opinions, and other statements in this presentation are the author's and not necessarily those of the sponsoring institution.
Overnights
• Grew cultures overnight at 37o while shaking• Used clear M9 media to supplement cells
Preparing Samples
• The next morning: took OD readings at 600nm and diluted cultures to OD values around 0.05• Grew cultures until all OD values fell within range of 0.5‐0.7• Dispensed samples and controls into 96/384 well plates
Plate Reader
• Added corresponding autoinducer molecules to each well and immediately began making measurements
• Measured both OD and fluorescence over course of 4 hours
3. Dimeractivatesinduciblepromoteronplasmid
4. PromoterdrivestranscriptionofGFP
Performed sensitivity analysis and optimized model parameters to qualitativelyrecapitulate biosensor performance
Sensitivity analysis showed that LasR concentration was sensitive to both transcriptionof mRNA and translation of LasR from mRNA
Analysis further showed that autoinducer concentrations was most sensitive toautoinducer degradation rates