Imperial/Cell by Date/Testing

From 2007.igem.org

(Difference between revisions)
m (Summary)
m
Line 19: Line 19:
__NOTOC__
__NOTOC__
= Cell by Date: Testing =
= Cell by Date: Testing =
-
 
-
<font color=red> Need to finish these pages and discussions </font>
 
==Summary==
==Summary==
Line 30: Line 28:
==Aims==
==Aims==
 +
 +
Having determined that our system worked at a basic level in our [https://2007.igem.org/Imperial/Cell_by_Date/Implementation implementation phase] we set out to ascertain a clearer picture of the performance of our system to ultimately reveal whether our design had realised our specifications.
==Results==
==Results==
Line 59: Line 59:
'''Other practical considerations:''' <br>
'''Other practical considerations:''' <br>
*Unfortunetly we have not been able to use a visible reporter and so have no idea whether the construct can in fact produce a visible signal. <br>
*Unfortunetly we have not been able to use a visible reporter and so have no idea whether the construct can in fact produce a visible signal. <br>
-
*In terms of lifespan we have had major problems with evaporation meaning that the lifespan of our system is limited to a few days. <br>
+
*In terms of lifespan we have had major problems with evaporation meaning that the lifespan of our system is limited to a few days as discussed in our packaging experiment below. <br>
===<font color=blue>Other Experiments</font>===
===<font color=blue>Other Experiments</font>===
Line 65: Line 65:
2. Packaging
2. Packaging
-
 
-
==Validation and Conclusion==
 

Revision as of 00:59, 27 October 2007



Cell by Date: Testing

Summary

Key results of our experiments are :

1.Operating Range : Our System Operates between 8-37 Degrees
2.Activation Energy : The activation energy of our system is 1.5kJ/mol
3.DNA Concentration : The optimum DNA concentration for our system is 2-4ug DNA

Aims

Having determined that our system worked at a basic level in our implementation phase we set out to ascertain a clearer picture of the performance of our system to ultimately reveal whether our design had realised our specifications.

Results

Investigating properties of system under isothermal scenarios

To estimate the properties of our system we looked at the evolution of the Fluorescence with time for experiments where temperature is carefully kept constant. (Figure 1).
General Behaviour:

  • In all experiment we can observe a linear growth of fluorescence corresponding to a steady rate of protein production.
  • This matches the claims of the manufacturer of the cell-free extract that the extract is optimised so that protein degradation is negligible.
Figr 1:Isothermal Data - Averages GFPmut3b molecules synthesised for pTet-GFPmut3b construct at different temperatures

Operating Range:

  • Our system seems to 'turn off' at around 4 degrees C.
  • At 8 degrees with have minimal expression which then increases to a substantial expression at 37 degrees.

Synthesis rate vs Temperature

  • Over the range of temperature considered here the rate increases with temperature.
  • To investigate the relation between synthesis rate and temperature we extracted the rate of synthesis of GFP for each experiment and plotted it against 1/T (Figure 2).
  • A strong linear correlation in the log plot supports an arrhenius type dependence on temperature with an activation energy of 1.5kJ/mol.
Fig 2: Arrhenius Plot

Investigating properties of system under isothermal scenarios

Fig 1.1:Average fluorescence over time for two samples at constant temperatures of 4&degC and 20°C and two with a temperature step (4-20°C and 20-4°C)

Other practical considerations:

  • Unfortunetly we have not been able to use a visible reporter and so have no idea whether the construct can in fact produce a visible signal.
  • In terms of lifespan we have had major problems with evaporation meaning that the lifespan of our system is limited to a few days as discussed in our packaging experiment below.

Other Experiments

1. DNA Concentration

2. Packaging



<< Implementation | Testing/Validation | Conclusions >>