MYN.U. P.A.L ...2011.igem.org/files/poster/Northwestern.pdf · Title: Microsoft PowerPoint - truly...

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