1. CELL BY DATE UPDATE 29.08.07 14:00

2. Presentation Outline CBD Background Project Approach What’s Done Lab Team Modelling Team Protocols Team What’s Next Q & A

3. Background Specifications Reports Thermal Exposure Accurately predict level of meat spoilage Use of cell-free systems BackgroundProject ApproachWhat’s DoneWhat’s NextQ & A CBD Reporter System Effects of temperature on gene expression Constitutive promoter Fluorescent protein (Example of a CBD DNA Construct)

4. Project Approach Phase 1 Aim: Initial Testing Build DNA constructs Initial Testing in vivo Initial Testing in vitro Generate equations for Modelling Phase 2 Aim: Characterization of Specific Construct Operating Range Effects of Temp. on Fluorescence vs. Time Lifespan Generate preliminary data for Modelling Phase 3 Aim: Testing/Validation of Predictive Model Accurate prediction of outcome Fluorescence vs. Time Lifespan Corroborate specs of device DsRed-Express & in- veso BackgroundProject ApproachWhat’s DoneWhat’s NextQ & A

5. Project Approach: Breakdown BackgroundDesign PhasesWhat’s DoneWhat’s NextQ & A Lab TeamModelling TeamProtocols Team Build / amplify DNA constructs Derive equations / Carry out simulations Gather / merge protocols Conduct Expts / Generate data for analysis Generation of predictive model

6. What’s Done Lab Team Cloning/Amplification pTet-GFP pT7-GFP Biobricks construct does not work well To be obtained from different sources pCI-GFP (Cancelled) Awaiting DsRed-Express BackgroundProject ApproachWhat’s DoneWhat’s NextQ & A

7. Modelling Team kFP : Function of Temperature. k is based on the promoter used as promoters take time to turn on. dFP : Function of System. May be considered to be a function of temperature as proteins degrade faster at higher temperatures. What’s Done BackgroundProject ApproachWhat’s DoneWhat’s NextQ & A

8. Modelling Team What’s Done BackgroundProject ApproachWhat’s DoneWhat’s NextQ & A

9. What’s Done Protocols Team BackgroundProject ApproachWhat’s DoneWhat’s NextQ & A Works in vivo & in vitro Tested @ 10 o C, 25 o C, 37 o C, 45 o C Does not work at 10 o C, 45 o C But when 10 o C left o/n at 25 o C, gene expression occurs Worked weakly in vivo & in vitro Very weak output cf. pTet To be obtained from different sources and tested

10. What’s Done Protocols Team BackgroundProject ApproachWhat’s DoneWhat’s NextQ & A (Gaps in data due to lab restrictions) (1 magnitude increase)

11. What’s Done Protocols Team BackgroundProject ApproachWhat’s DoneWhat’s NextQ & A (Not working well) (Difference in expression in vivo)

12. What’s Done BackgroundProject ApproachWhat’s DoneWhat’s NextQ & A Phase 1 Aim: Initial Testing Build DNA constructs Initial Testing in vivo Initial Testing in vitro Generate Equations for Modelling Phase 2 Aim: Characterization of Specific Construct Operating Range Effects of Temp. on Fluorescence vs. Time Lifespan Generate preliminary data for Modelling Phase 3 Aim: Testing/Validation of Predictive Model Accurate prediction of outcome Fluorescence vs. Time Lifespan Corroborate specs of device DsRed-Express & in- veso

13. What’s Next Timeline BackgroundProject ApproachWhat’s DoneWhat’s NextQ & A Week 8 Week 9 Week 10 Phase 2 [GFP] & δ[GFP] curves Homemade cell extract Phase 2 Effects of temperature Gentle / steep gradients Preliminary data analysis Phase 3 Suitable temp. step increases Refine predictive model Phase 4? Proper documentation Substitute GFP w/ DsRed- Express In-veso gene expression Packaging Methods

14. What’s Next BackgroundProject ApproachWhat’s DoneWhat’s NextQ & A

15. BackgroundProject ApproachWhat’s DoneWhat’s NextQ & A