From 2007.igem.org

Revision as of 23:55, 25 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

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.

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:

1. Rate of GFP synthesis of 100nM
2. Transfer Function.

Comparison between in vivo and in vitro for rate of GFPmut3b at 100nM AHL. The in vivo chassis was MG1655 and the in vitro chassis was commcerial S30 cell extract. *in vivo has a maximal rate of 400-500 molecules of GFP synthesised per second per cell. In addition the in vivo chassis reaches a steady state around 30minutes. *in vitro has the equivalent of 220 molecules of GFP synthesised per second per cell equivalent, the cell equilavent being based upon the normalization of DNA plasmids. 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. The reason why the in vitro chassis never reaches a steady state is because the in vitro chassis has limited energy and metabolites available this is unlike the in vivo chassis that has media available. 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.

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: