IGEM:IMPERIAL/2007/new pages/testing validation

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Contents

Results


The results from the final testing are shown in the diagram above, the protocol and results can be found on the following links....

The fluorescence was converted using our calibration curve, for an explanation see conversion of units page. The results show us the following:

  • The output of GFPmut3b increases with input of AHL
  • The system is sensitive to a range of 5-1000nM AHL
  • The GFPmut3b molecules synthesis stops at ~300minutes. This could be due to steady state or due to no synthesis of GFPmut3b. It is known not to be steady state because the degradation experiment(link) proved degradation is negligible. Interestingly this time is independent of the GFPmut3b molecules produced, showing that the LuxR under the control of pTet is the major source of energy consumption. This highlights the advantages of using the construct 2 pLux-GFPmut3b that does not have the energetic burden of producing LuxR

In addition the results in vitro have been compared to the work on BBa_F2620(pTet-LuxR-pLux-GFPmut3b in vivo which is the same as the construct 1 used for infecter detector. To do this we investigated the comparison between in vitro and in vivo.



Normalise the in vitro on the plasmids to give a platform for comparison:

  • In Vitro - 4µg of DNA was added which for pTet-LuxR-pLux-GFPmut3b is 904823007 plasmids
  • In Vivo - Each cell has ~30 plasmids per cell

To compare we normalised the data of in vitro GFPmut3b molecules synthesised per 30 plasmids to allow some comparison to the in vivo data.

Rate of GFPmut3b Synthesis for 100nM AHL


Comparison between in vivo and in vitro for rate of GFPmut3b at 100nM AHL. The rate for in vitro and in vivo was taken for the maximum rate for each chassis.  *in vivo has a maximal rate of 400-500 molecules of GFP synthesised per second per cell. The in vivo reaches steady state ~30minutes. *in vitro has the equivalent of 220 molecules of GFP synthesised per second per cell equivalent. This is based upon the normalization on DNA plasmids. The in vitro chassis decreases in rate of synthesis after 90 minutes and keeps decreasing until rate is zero at around 360 minutes
Comparison between in vivo and in vitro for rate of GFPmut3b at 100nM AHL. The rate for in vitro and in vivo was taken for the maximum rate for each chassis.
*in vivo has a maximal rate of 400-500 molecules of GFP synthesised per second per cell. The in vivo reaches steady state ~30minutes.
*in vitro has the equivalent of 220 molecules of GFP synthesised per second per cell equivalent. This is based upon the normalization on DNA plasmids. The in vitro chassis decreases in rate of synthesis after 90 minutes and keeps decreasing until rate is zero at around 360 minutes

Transfer Function


The graph above shows the transfer function of AHL input vs rate of GFP synthesis output. The blue line on in vivo corresponds to the range of AHL on in vitro *in vitro shows a similar shape to the in vivo transfer function, however rate of GFP synthesis  lower in the in vitro chassis. e.g. for 1000nM the rate in vivo is ~450 GFP molecules per sec per cell, in vitro has an equivalent value of 220 GFP molecules per second. *The in vitro chassis looks as if the rate is very low for low AHL inputs being <10 molecules of GFP per second.
The graph above shows the transfer function of AHL input vs rate of GFP synthesis output. The blue line on in vivo corresponds to the range of AHL on in vitro
*in vitro shows a similar shape to the in vivo transfer function, however rate of GFP synthesis lower in the in vitro chassis. e.g. for 1000nM the rate in vivo is ~450 GFP molecules per sec per cell, in vitro has an equivalent value of 220 GFP molecules per second.
*The in vitro chassis looks as if the rate is very low for low AHL inputs being <10 molecules of GFP per second.

Summary

Below is list of which of the orginial Specifications that our infecter detector achieved:

Property
Value
Achievements
Inputs
System must be sensitive to AHL concentration between 5-50nM
Sensitive to 5-1000nM
Outputs
System must give a visual signal if bacteria is present
Future work - Using Stronger fluorescent protein such as DsRed express
Response Time
System needs to have a response time under 3 hour
Systems responds <30minutes
Operating Conditions
System must operate within temperature 20-30°C
System works at 25°C
Health & Safety
System Must not be living replicating bacteria, and in any way harmful or infectious.
Cell Free in vitro chassis
Lifespan
System must have a shelf life of 7 days
Can be stored in freezer for prolonged periods
Packaging
System must be portable and convenient to use
Future Work - Using our chassis in a spray


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