|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.
has a maximal rate of 400-500 molecules of GFP synthesised per second per cell. The in vivo
reaches steady state ~30minutes.
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
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
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.
in vitro chassis looks as if the rate is very low for low AHL inputs being <10 molecules of GFP per second.
Below is list of which of the orginial Specifications that our infecter detector achieved:
||System must be sensitive to AHL concentration between 5-50nM
||Sensitive to 5-1000nM
||System must give a visual signal if bacteria is present
||Future work - Using Stronger fluorescent protein such as DsRed express
||System needs to have a response time under 3 hour
||Systems responds <30minutes
||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
||System must have a shelf life of 7 days
||Can be stored in freezer for prolonged periods
||System must be portable and convenient to use
||Future Work - Using our chassis in a spray