Leukemia Components of the bone marrow Eosinophil Myelocyte - PowerPoint PPT Presentation

Leukemia Every 4 minutes a new person is diagnosed Every 10 minutes somebody dies 10% of all leukemia patients are under 20

Team 2010 BIOTEC Dresden Svea & Mareike ricks ensor iology ynthetic

Leukemia Components of the bone marrow Eosinophil Myelocyte Normoblast Erythrocyte Fat

Current Leukemia Diagnosis State of the art CD33+ cell Anti-CD33 FACS

Desired Features of SensorBricks Sensitivity Simplicity Modularity

SensorBricks - ToolBox Protein A from Staphyloccocus aureus CD33+ cell Domain B of Protein A

SensorBricks - ToolBox Quorum Sensing System from Vibrio fischeri SAM LuxI LuxR AHL

SensorBricks - ToolBox

SensorBricks in Real Life Mix patients blood anti-CD33 + cells Fusionprotein

SensorBricks in Real Life Mix Antibody- patients blood Fusionprotein + anti-CD33 + cells blood sample Fusionprotein

SensorBricks in Real Life Mix Antibody- Take SN patients blood anti-CD33 + Fusionprotein + containing cells Fusionprotein blood sample AHL

SensorBricks in Real Life Detect Mix Antibody- Take SN add sensor patients blood fluorescent anti-CD33 + Fusionprotein + containing signal cells Fusionprotein blood sample AHL pTet LuxR pLuxR YFP

Experimental Pipeline Build Test Test Establish Simulate Detection SensorBricks SensorBricks Leukemia AHL sender System receiver sender Test

Results Build Test Test Establish Simulate Detection SensorBricks SensorBricks Leukemia AHL sender System receiver sender Test

Building a Detection System pTet LuxR pLuxR YFP

Building a Detection System Building a Detection system pTet RFP pTet LuxR pLuxR YFP RFC 78

SensorBricks in Real Life pTet RFP pTet LuxR pLuxR YFP

SensorBricks in Real Life pTet RFP pTet LuxR pLuxR YFP

Results Build Test Test Establish Simulate Detection SensorBricks SensorBricks Leukemia AHL sender System receiver sender Test

Results Build Test Test Establish Simulate Detection SensorBricks SensorBricks Leukemia AHL sender System receiver sender Test

Test SensorBricks Receiver The fluorescence signal is proportional to the AHL input [AHL] in nM 26,00 2000 24,00 1000 Fluorescence (YFP/RFP) 500 22,00 200 100 20,00 50 18,00 10 1 16,00 0,1 0,01 14,00 0 12,00 0 20 40 60 80 100 120 140 Time in minutes

Test SensorBricks Receiver Direct correlation of fluorescence to [AHL] after 2 hours 22 Fluorescence (YFP/RFP) 20 18 16 14 12 10 0 500 1000 1500 2000 AHL concentration in nM

Results Build Test Test Establish Simulate Detection SensorBricks SensorBricks Leukemia AHL sender System receiver sender Test

Results Build Test Test Establish Simulate Detection SensorBricks SensorBricks Leukemia AHL sender System receiver sender Test

Simulate AHL Sender Proof of Principle: Cocultivation Assay Sender expressing LuxI pTet LuxI pTet RFP pTet LuxR pLuxR YFP Receiver detecting AHL

Simulate AHL Sender Low amounts of LuxI lead to a detectable induction 140 120 Fluorescence (YFP/RFP) 100 80 60 40 20 0 Sender Receiver +500 nM AHL 20:1 100:1 1000:1 10000:1 Ratio Receiver : Sender

Results Build Test Test Establish Simulate Detection SensorBricks SensorBricks Leukemia AHL sender System receiver sender Test

Future Outlook Build Test Test Establish Simulate Detection SensorBricks SensorBricks Leukemia AHL sender System receiver sender Test

Future Outlook Build Test Test Establish Simulate Detection SensorBricks SensorBricks Leukemia AHL sender System receiver sender Test

Potential of SensorBricks Amplification pTet RFP pTet LuxR pLuxR LuxI YFP

Potential of SensorBricks Amplification pTet RFP pTet LuxR pLuxR LuxI YFP Pigments http://2009.igem.org/Team:Cambridge

Potential of SensorBricks Amplification pTet RFP pTet LuxR pLuxR LuxI YFP Pigments Modularity Multiple Detection

Thanks to: Andy Oates, Annelie Oswald, Marko Storch, Petra Schwille, Johnson Madrid, Kaj Bernhardt, Michael Bachmann BIOTEC Dresden, MPI-CBG Dresden, TU Dresden

FACS Facts Feature FACS SensorBricks Detection Limit 1 cell in 10 000 cells 1 sender cell in 10 000 receiver cells Price (Hardware) in US$ ~ 50 000 ~ 200 000 Total test time 3 h 4-5 h