Imperial/Wet Lab/Results/ID3.1

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The idea behind using molecules of GFPmut3b is to try to standardise our units for the chassis and normalise our data based upon the number of DNA plasmids present in our system. This idea is similar to that for the F2620 which uses the units GFP molecules synthesized per second per cell. Trying to normalise this data with the plasmids helps to make our data independent of the DNA used in this assay, i.e. for other constructs tested and characterized in vitro.
The idea behind using molecules of GFPmut3b is to try to standardise our units for the chassis and normalise our data based upon the number of DNA plasmids present in our system. This idea is similar to that for the F2620 which uses the units GFP molecules synthesized per second per cell. Trying to normalise this data with the plasmids helps to make our data independent of the DNA used in this assay, i.e. for other constructs tested and characterized in vitro.
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There are several key parameters that can be extracted from the data. It looks as if our system becomes saturated to AHL ( the input) above 100nM of AHL, it can be seen that there is little difference between 1000nM and 100nM. If we assume the GFP synthesis 1000nM to be the maximum then the switch point , defined when the output is 50% of the max, is around 20nM of AHL.
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Figure 1.1 shows;
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*Functional at 25<sup>o</sup>C
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The response time is a key characteristic however from this data it is difficult to get an accurate value. The problem is that because the temperature was regulated sampling had to be restricted to every 30 minutes, meaning our best approximation is <30minutes.
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*The output of '''GFPmut3b increases with input of AHL'''
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*The system is sensitive to a range of '''5-1000nM AHL'''
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The sensitivity of the chassis and construct can also be deduced for the range of AHL concentrations that we looked at. The sensitivity to AHL is <5nM.
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*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 [[Imperial/Wet_Lab/Results/Res1.4| degradation experiment]] proved degradation is negligible. Interestingly this time at which synthesis stops is independent of the GFPmut3b molecules produced as all the [AHL] level off at the same time. This shows that the LuxR under the control of pTet is the major source of energy consumption. The pTet is a very strong promoter and is a big consumer of the energy of the chassis. This highlights the advantages of using the construct 2 [http://partsregistry.org/Part:BBa_J37032 pLux-GFPmut3b] that does not have the energetic burden of producing LuxR under pTet. <br><br>
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Figure 1.2 shows us the following:
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*The shape of the '''Transfer function''' shows a linear range of response between 5nM and 100nM AHL. This defines the thresholds of response.
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*The '''lower threshold of response''' is the AHL concentration that the construct will respond
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*The '''upper threshold of response''' is the value of AHL the system is saturated and increasing AHL will not increase the rate of GFP synthesis.<br><br>
==Conclusion==
==Conclusion==

Revision as of 01:36, 27 October 2007


In vitro Testing of pTet-LuxR-pLux-GFPmut3b Construct with varying concentrations of AHL

Aims

To characterise the [http://partsregistry.org/Part:BBa_T9002 pTet-LuxR-pLux-GFPmut3b] construct in vitro. The characterisation is based upon giving inputs of varying AHL concentration and measuring the fluorescent output.


Materials and Methods

Link to the Protocols

Results


Figure 1.1 pTet-LuxR-pLux-GFPmut3b in vitro - The graph shows the concentration of GFPmut3b against time for varying levels of AHL

Figure 1.2 pTet-LuxR-pLux-GFPmut3b in vitro - The graph shows the a titration curve of AHL vs GFPmut3b after 360minutes


Controls:

  • Negative Control- pTet-LuxR-pLux-GFPmut3b with no AHL
  • Negative Control.2.- pLux with 1000nM AHL


Constants:

  • Temperature - 25°C
  • Volume - 60ul
  • Sampling on same plate and every 30 minutes

Raw Data
File:AHL testing Final1.xls

Discussion

The results from the experiment were of fluorescence against time for varying levels of AHL. Using our calibration curves we managed to convert the fluorescence into concentration of GFPmut3b produced (figure 1.1) and also into Molecules of GFPmut3b synthesised per plasmid (figure 1.2). See the how the calibration curves allowed us to do this by following this link to converting units.

The idea behind using molecules of GFPmut3b is to try to standardise our units for the chassis and normalise our data based upon the number of DNA plasmids present in our system. This idea is similar to that for the F2620 which uses the units GFP molecules synthesized per second per cell. Trying to normalise this data with the plasmids helps to make our data independent of the DNA used in this assay, i.e. for other constructs tested and characterized in vitro.

Figure 1.1 shows;

  • Functional at 25oC
  • 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 proved degradation is negligible. Interestingly this time at which synthesis stops is independent of the GFPmut3b molecules produced as all the [AHL] level off at the same time. This shows that the LuxR under the control of pTet is the major source of energy consumption. The pTet is a very strong promoter and is a big consumer of the energy of the chassis. This highlights the advantages of using the construct 2 [http://partsregistry.org/Part:BBa_J37032 pLux-GFPmut3b] that does not have the energetic burden of producing LuxR under pTet.

Figure 1.2 shows us the following:

  • The shape of the Transfer function shows a linear range of response between 5nM and 100nM AHL. This defines the thresholds of response.
  • The lower threshold of response is the AHL concentration that the construct will respond
  • The upper threshold of response is the value of AHL the system is saturated and increasing AHL will not increase the rate of GFP synthesis.

Conclusion

These are the characteristics we have extracted

Switch Point= ~20nM AHL
Response Time= <30 minutes
Sensitivity = <5nM AHL

In terms of the specification of the Infector Detector Application the chassis and construct is sensitive enough.

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