From 2007.igem.org

Revision as of 10:30, 24 October 2007

• Home
  • Site Map
  • Meet the Team
  • Fun with iGEM
  • IcGEMs@OpenWetware
  • Acknowledgements
• Infector Detector
  • Introduction
  • Specification
  • Design
  • Modelling
  • Implementation
  • Testing/Validation
  • F2620 Comparison
  • Conclusion
• Cell by Date
  • Introduction
  • Specification
  • Design
  • Modelling
  • Implementation
  • Testing/Validation
  • Conclusion
• Cell-Free
  • What is Cell-Free?
  • Cell-Free Vs. Cell
  • Our Contributions
  • Characterisation
  • Applications
• Wet Lab
  • Lab Notebook
  • Protocols & Results
  • DNA Constructs
• Dry Lab
  • Modelling
  • Data Analysis
  • Software

Infector Detector: Testing

Aims

The sensitivity of the system is to be determined through this experiment. To do this, we induce the system with known concentrations of AHL. We then record the change in GFP, such that we can calculate the rate of GFP production relative to concentration of AHL in solution.

• To determine the rate of protein production relative to concentration of AHL
• To determine the response time relative to concentration of AHL
• To determine the steady state response of protein relative to concentration of AHL

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 [http://partsregistry.org/Part:BBa_J37032 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 [http://partsregistry.org/Part:BBa_F2620 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 [http://partsregistry.org/Part:BBa_T9002 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

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:

Testing and Results

Determine AHL sensitivity

We first induced the system with varying concentrations of AHL to determine the output of the system. From the results, we were able to determine that the sensitivity range of the system was indeed between 5-50 nM, which is what was specified earlier. We can thus calibrate the system to give a visual output at this range of AHL concentrations.

Transient reponse of the system at different AHL concentrations. There is a clear trend that with increasing amount of AHL concentration, the GFP output of the system also increases.

The transfer function of our system - with the output of GFP at 3 hours af varying AHL inputs. This curve behaves similiarly as it would in vivo, with the highest sensitivity at around 5-50nM.

Discussion and Validation