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CELL BY DATE UPDATE29.08.0714:00
Presentation Outline
CBD Background Project Approach What’s Done
Lab Team Modelling Team Protocols Team
What’s Next Q & A
Background
Specifications Reports Thermal Exposure Accurately predict level of meat
spoilage Use of cell-free systems
Background Project Approach What’s Done What’s Next Q & A
CBD Reporter System Effects of temperature on gene
expression Constitutive promoter Fluorescent protein
(Example of a CBD DNA Construct)
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
Background Project Approach What’s Done What’s Next Q & A
Project Approach: Breakdown
Background Design Phases What’s Done What’s Next Q & A
Lab Team Modelling Team Protocols Team
Build / amplifyDNA constructs Derive equations /
Carry out simulations
Gather / mergeprotocols
Conduct Expts /Generate data for
analysisGeneration of predictive model
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
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.
What’s Done
Background Project Approach What’s Done What’s Next Q & A
Modelling Team
What’s Done
Background Project Approach What’s Done What’s Next Q & A
What’s Done
Protocols Team
Background Project Approach What’s Done What’s Next Q & A
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
What’s Done Protocols Team
pTet in Vitro - 10 Degrees
0
5000
10000
15000
20000
25000
30000
00:00 01:00 02:00 03:00 04:00 22:00 05:30
Sample
Fluo
resc
ence
A1 - pTet in Vitro
A1 - pTet in Vitro
A1 - pTet in Vitro
GFP dilution
Cell extract only
Blank control
Blank control
Blank control
Background Project Approach What’s Done What’s Next Q & A
pTet Vitro - 37 degrees
0
5000
10000
15000
20000
25000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Hours (aprx)
Flu
ore
sc
en
ce
pTet 1
pTet 2
GFP
Neg. Control
(Gaps in data due to lab restrictions)(1 magnitude increase)
What’s Done Protocols Team
pT7 Vitro 37oC
0
5000
10000
15000
20000
25000
30000
00:00 00:30 01:00 01:30 02:00 02:30 03:00 03:30 04:00 22:00 02:30 05:30
Sample
Fluore
scen
ce
A1 - pT7 in Vitro
A2 - pT7 in Vitro
A3 - pT7 in Vitro
Cell extract only
GFP dilution
Blank control
Blank control
Blank control
Background Project Approach What’s Done What’s Next Q & A
Fluorescence Vs Time
0
20000
40000
60000
80000
100000
120000
140000
0.000 20.000 40.000 60.000 80.000 100.000
Time (sec)
Fluo
resc
ence
A1 pTet
A2 pTet
A3 pTet
A5 LB-Media
A6 LB-Media
A7 LB-Media
B1 pT7-GFP
B2 pT7-GFP
B3 pT7-GFP
B4 LB-Media + Non expressive culture
B5 LB-Media + Non expressive culture
B6 LB-Media + Non expressive culture
C1 Diluted GFP
C2 Diluted GFP
C3 Diluted GFP
H10 Blank Control
H11 Blank Control
H12 Blank Control
(Not working well)(Difference in expression in
vivo)
What’s Done
Background Project Approach What’s Done What’s Next Q & 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
What’s Next
Timeline
Background Project Approach What’s Done What’s Next Q & 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
Q & A
Background Project Approach What’s Done What’s Next Q & A