Arabinose SensorHSL AmplifierLevels of Abstraction - System
Arabinose PromoterHSL Sender/RecieverHSL SenderCFPYFPLevels of Abstraction - Devices
Levels of Abstraction - Parts
Expected OutputExpanding YFP ring surrounding CFP circle shows signal propogationMedia plate w/ bacterial lawn
ArabinoseMechanism
HSLMechanism
HSLHSLMechanism
Construction2134 partsFinal
Construction Results2134 partsFinal
FUTURE WORK
Check construct via sequencing Reconstruct if necessary
Simone MacmilDurga P Sarvepalli
4 November 2006Detecting small molecule signaling using phosphorylation dependent mechanism in E.coli
SMALL MOLECULES Smallest part of pure chemical substance that retains its structure and properties.Play an important role in multiple signaling mechanisms
APPLICATIONS OF SMALL MOLECULES IN BIOLOGYSmall molecule drugsBiopolymersSynthetic peptidesPrimers
NEED FOR SMALL MOLECULE DETECTIONUnderstand molecule protein interactionsEffect of molecules on the viability of cellsGenetic changes caused by molecules used in molecular therapyDrug discovery
CURRENTLY AVAILABLE TECHNIQUESAnalytical: NMR, Western blot, Spectrophotometry, Chromatography, ELISASmall molecule protein interactions : Nanowire sensors ( Wang et al , PNAS 2005;102;3208-3212)Disadvantages: whole cells cannot be usedUsing micro channels whole cells can be used and less amount of reagents requiredMicro channels can also be customized according to the design of the experiment and allows studies to be conducted under flow
FABRICATION OF MICROFLUIDIC CHANNELPDMS - Poly dimethyl siloxane
MICROFLUIDIC CHANNEL DIMENSIONSChannel width : 200m depth : 50 m
DEVICEInduction
METHODSTransformationParts from the registryRestriction enzyme digestionSequential ligation to create constructSequence transformantsLigate into pUC Grow selected transformants in micro channels
RESULTSAspartate detectionMicrofluidic channelCells grown in Minimal Media lacking aspartate and induced with IPTGCells fluoresce after 30 min in the presence of LB
Movement of E.coli in the channelRESULTS
FUTURE WORK
Improvise microchannels
- Study flow of bacteria under various conditions - chemotaxis, sensitivity of bacteria in the gut to pH, temperature.
Detect low concentration of small molecules formed during bio-industrial production.
-Design appropriate sensors for detection (small peptides and sugars)
ACKNOWLEDGEMENTSDr. Bruce RoeDr. Matthias NollertDr. David SchmidtkeDr. Fares NazarDr. Randall HewesDoug WhiteSandra Bryant
