CELL BY DATE UPDATE

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 Background Project Approach What’s Done What’s Next Q & A • Specifications • Reports Thermal Exposure • Accurately predict level of meat spoilage • Use of cell-free systems • CBD Reporter System • Effects of temperature on gene expression • Constitutive promoter • Fluorescent protein (Example of a CBD DNA Construct)

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

5. Project Approach: Breakdown Background Design Phases What’s Done What’s Next Q & A Lab Team Modelling Team Protocols Team Build / amplify DNA constructs Derive equations / Carry out simulations Gather / mergeprotocols Conduct Expts / Generate data for analysis Generation of predictive model

6. What’s Done Background Project Approach What’s Done What’s Next Q & A • 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

7. What’s Done Background Project Approach What’s Done What’s Next Q & A • 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.

8. What’s Done Background Project Approach What’s Done What’s Next Q & A • Modelling Team

9. What’s Done Background Project Approach What’s Done What’s Next Q & A • Protocols Team • Works in vivo & in vitro • Tested @ 10oC, 25oC, 37oC, 45oC • Does not work at 10oC, 45oC • But when 10oC left o/n at 25oC, 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 Background Project Approach What’s Done What’s Next Q & A • Protocols Team (Gaps in data due to lab restrictions) (1 magnitude increase)

11. What’s Done Background Project Approach What’s Done What’s Next Q & A • Protocols Team (Not working well) (Difference in expression in vivo)

12. What’s Done Background Project Approach What’s Done What’s Next Q & A Phase 1 Phase 2 Phase 3 Aim: Initial Testing • Build DNA constructs • Initial Testing in vivo • Initial Testing in vitro • Generate Equations for Modelling Aim: Characterization of Specific Construct • Operating Range • Effects of Temp. on • Fluorescence vs. Time • Lifespan • Generate preliminary data for Modelling 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 Background Project Approach What’s Done What’s Next Q & A Timeline • Phase 2 • [GFP] & δ[GFP] curves • Homemade cell extract • Phase 3 • Suitable temp. step increases • Refine predictive model Week 8 Week 10 Week 9 • Phase 4? • Proper documentation • Substitute GFP w/ DsRed-Express • In-veso gene expression • Packaging Methods • Phase 2 • Effects of temperature • Gentle / steep gradients • Preliminary data analysis

14. Q & A Background Project Approach What’s Done What’s Next Q & A