Imperial/Infector Detector/F2620 Comparison

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=Comparison to F2620=
=Comparison to F2620=
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For further analysis the results of our ''in vitro'' testing have been compared to the work on [http://partsregistry.org/Part:BBa_F2620 BBa_F2620]('''pTet-LuxR-pLux-GFPmut3b''') ''in vivo'' which is the same as the construct 1 that was used for our infecter detector system. The motivation of the comparison is to see how this construct will respond in different chassis. To do this we investigated a standard unit s to allow the comparison between ''in vitro'' and ''in vivo.''
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For further analysis the results of our ''in vitro'' testing have been compared to the work ''in vivo'' on [http://partsregistry.org/Part:BBa_F2620 BBa_F2620]('''pTet-LuxR-pLux-GFPmut3b'''), the construct being the same as our construct 1 for infecter detector. The motivation of the comparison is to see how this construct will respond in different chassis. To do this we investigated a standard unit s to allow the comparison between ''in vitro'' and ''in vivo.''
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Normalise the ''in vitro'' on the number of plasmids to give a platform for comparison:
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The basis for comparison is to normalise the ''in vitro'' chassis on the number of plasmids to give a platform for comparison:
*''In Vitro'' - 4&micro;g of DNA was added which for [http://partsregistry.org/Part:BBa_T9002 '''pTet-LuxR-pLux-GFPmut3b'''] is 904823007 plasmids
*''In Vitro'' - 4&micro;g of DNA was added which for [http://partsregistry.org/Part:BBa_T9002 '''pTet-LuxR-pLux-GFPmut3b'''] is 904823007 plasmids
*''In Vivo'' - Each cell the plasmids number was estimated at 30 per cell
*''In Vivo'' - Each cell the plasmids number was estimated at 30 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.   
To compare we normalised the data of ''in vitro'' '''GFPmut3b molecules synthesised per 30 plasmids''' to allow some comparison to the ''in vivo'' data.   
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Of particular interest was to compare the '''Rate of GFP synthesis''' of 100nM and the '''Transfer Function'''.
===Rate of GFPmut3b Synthesis for 100nM AHL===
===Rate of GFPmut3b Synthesis for 100nM AHL===
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Revision as of 23:10, 25 October 2007



Comparison to F2620

For further analysis the results of our in vitro testing have been compared to the work in vivo on [http://partsregistry.org/Part:BBa_F2620 BBa_F2620](pTet-LuxR-pLux-GFPmut3b), the construct being the same as our construct 1 for infecter detector. The motivation of the comparison is to see how this construct will respond in different chassis. To do this we investigated a standard unit s to allow the comparison between in vitro and in vivo.



IC2007 ,,BF stage1 construct.PNG

The basis for comparison is to normalise the in vitro chassis on the number of plasmids to give a platform for comparison:

  • In Vitro - 4µg of DNA was added which for [http://partsregistry.org/Part:BBa_T9002 pTet-LuxR-pLux-GFPmut3b] is 904823007 plasmids
  • In Vivo - Each cell the plasmids number was estimated at 30 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.

Of particular interest was to compare the Rate of GFP synthesis of 100nM and the Transfer Function.

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

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.

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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