The key differences are between the chassis is the rate of GFP synthesis which is lower in the ''in vitro'' chassis e.g. for 1000nM of AHL the rate of GFP synthesis ''in vivo'' is ~450 GFP molecules per sec per cell,'' ''in vitro'' has an equivalent value of 220 GFP molecules per second.<br>
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*'''Shape of Transfer Function'''- a sigmoidal shaped transfer function with a step linear region flanked by a lower threshold and upper threshold is seen for both chassis. This indicated that the characteristic response of the construct are independent of the chassis for a given strain i.e both of these ''in vitro'' and ''in vivo'' chassis are from ''E.coli''.
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*'''Rate of GFP Synthesis''' - The rate of synthesis is greater in the ''in vitro'' chassis e.g. for 1000nM AHL the rate is 1400 GFP molecules synthesis per second per cell equivalent for ''in vitro'' and 500 GFP molecules synthesis per second per cell. This makes sense because the commercial E.coli cell extract we use is optimised for protein synthesis.
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The shape of the transfer function is very similar for both chassis, both begin to saturate at around 1000nM of AHL and the threshold of sensitivity is around 1nM AHL. It is very surprising that a construct works so similar in different chassis, showing the affect of the chassis is minimal to the constructs behavior.
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*'''Upper Threshold''' - The upper threshold of response looks as if it occurs at ~1000nM for both chassis. The saturation is due to the saturation of expression equipment, meaning that the system cannot respond to any increase to AHL.
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*'''Lower Threshold''' - The lower threshold of response is 1nM for ''in vivo'', however because of the lack of data points we had to extrapolate this for ''in vitro''. From the shape of the ''in vitro'' data we collected it looks like the threshold could be at a higher concentration to AHL, meaning that the ''in vitro'' chassis is less sensitive to lower AHL concentrations. However without these data points it is difficult to define this threshold accurately.
We thought to compare our in vitro characterisation to the characterisation of [http://partsregistry.org/Part:BBa_F2620 F2620] in vivo with the aim to highlight some of the differences between the chassis and investigate how the construct's characteristics may change between them. The [http://partsregistry.org/Part:BBa_F2620 F2620] is an ideal construct for comparison because of its detailed characterisation in vivo. The construct is the same as the construct 1 that was used for infecter detector, pTet-LuxR-pLux-GFPmut3b. The key results of the comparison were:
The creation of a new unit to allow comparison between in vitro and in vivo chassis.
That although we are changing the E.coli chassis from in vivo to in vitro the construct's characteristic response is independent of the chassis.
These are exciting findings, revealing the potential for the exploration of new chassis and the ability to use constructs in an exchangable manner.
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 200 per cell
To compare we normalised the data of in vitroGFPmut3b molecules synthesised per 200 plasmids to allow some comparison to the in vivo data.
Of particular interest was to compare the:
Rate of GFP synthesis of 100nM AHL
Transfer Function.
Comparison between in vivo and in vitro for rate of GFPmut3b synthesis for 100nM AHL. The in vivo chassis used was the bacterial strain MG1655 and the in vitro chassis was Promega Commercial S30 Cell Extract(60µl)
The graph shows the following:
in vivo has a maximal rate of 400-500 molecules of GFP synthesised per second per cell. In addition the rate reaches a steady state after around 30minutes and maintains it for the duration of the testing.
in vitro has the equivalent of 1400 molecules of GFP synthesised per second per cell equivalent, the cell equilavent being based upon the normalization of DNA plasmids. Interestingly the in vitro chassis does not reach a steady state, in fact it decreases in rate of synthesis after 90 minutes and keeps decreasing until rate is zero at around 360 minutes.
Key Difference:
GFP Synthesis - The life of synthesis of in vitro is about 90 minutes, compared to in vivo that has a life span of days. The differences is that in vitro has a burst of expression giving a high but short rate of GFP synthesis. In vivo is a slower rate but more prolonged rate of synthesis.
Intial Rate of Synthesis - The initial rate of GFP synthesis is slower for in vitro. This is thought to be due to LuxR levels, which in vivo are at steady state but in vitro has to produce the LuxR upon addition of DNA.
Interestingly the values of rate of synthesis are in the same order magnitude of hundreds, this suggesting that the normalisation we are using to compare these chassis is valid.
The graph above shows the transfer function of [AHL] input vs rate of GFP synthesis output. The graph shows the follwoing:
in vivo(blue line) max rate of synthesis which is the steady state that is reached after about 30 minutes
in vitro(red line) max rate of synthesis which is the rate between 60 and 90 minutes which is the maximum rate before the energy limitations of the system cause the rate to drop. The dotted red line represents the expected transfer function for the lower AHL concentrations that we did not collected data points for.
Key Difference:
Shape of Transfer Function- a sigmoidal shaped transfer function with a step linear region flanked by a lower threshold and upper threshold is seen for both chassis. This indicated that the characteristic response of the construct are independent of the chassis for a given strain i.e both of these in vitro and in vivo chassis are from E.coli.
Rate of GFP Synthesis - The rate of synthesis is greater in the in vitro chassis e.g. for 1000nM AHL the rate is 1400 GFP molecules synthesis per second per cell equivalent for in vitro and 500 GFP molecules synthesis per second per cell. This makes sense because the commercial E.coli cell extract we use is optimised for protein synthesis.
Upper Threshold - The upper threshold of response looks as if it occurs at ~1000nM for both chassis. The saturation is due to the saturation of expression equipment, meaning that the system cannot respond to any increase to AHL.
Lower Threshold - The lower threshold of response is 1nM for in vivo, however because of the lack of data points we had to extrapolate this for in vitro. From the shape of the in vitro data we collected it looks like the threshold could be at a higher concentration to AHL, meaning that the in vitro chassis is less sensitive to lower AHL concentrations. However without these data points it is difficult to define this threshold accurately.
