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Revision as of 23:35, 26 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

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Infector Detector: Conclusion

The main achievements of the Infector Detector project:

• Extensive modelling of the two potential constructs for Infector Detector
• Purification of GFPmut3b to allow construction of a calibration curve
• Detailed characterisation of construct 1 in vitro using a calibration curve to find rate of GFP synthesis
• Creation of a standard unit to allow comparison between in vitro and in vivo

The table below summarises our Infector Detector system in the context of the original specifications:

Property	Specification	Achievements
Inputs	System must be sensitive to AHL concentration between 5-50nM	Sensitive to 5-1000nM
Outputs	System must give a visual signal if bacteria is present	Future work - Using Stronger fluorescent protein such as DsRed express
Response Time	System needs to have a response time under 3 hour	Systems responds <30minutes
Operating Conditions	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
Shelf-life	System must have a shelf life of 7 days	Can be stored in freezer for prolonged periods
Packaging	System must be portable and convenient to use	Future Work - Using our chassis in a spray

Battle a spectrum of infections

The great potential of Infector detector is that it is not limited to just one infection. Adding sensitivity to AHL originating from biofilms is just the beginning. By tweaking the internal mechanisms of the construct, Infector Detector can be used to battle a range of catheter-related bacteremias. For example by using a construct that recognises AI-2¹ , we can detect the presence of Klebsiella pneumoniae, a pathogenic bacterium ranked second to E. coli for urinary tract infections in older persons.

Added control - Construct 2

The main advantage of using construct 2 is that it provides an additional control mechanism for our detector meaning that you can tweak the detector sensitivity.
Going into deeper detail, construct 1 can produce LuxR as soon as it is activated. LuxR's presence is necessary for the formation of AHL-LuxR complex and the subsequent activation of pLux (leading to GFP production). Construct 2 on the other hand does not have a LuxR producing part. It relies on the user to add the necessary LuxR to form the binding complex. This control over LuxR can thus act as a sort of attenuator to the sensitivity of Infector Detector.
Having little LuxR present, will form very little binding complex with AHL and thus the sensitivity will decrease significantly. Saturating the detection compound with LuxR will maximise the sensitivity. Briefly, if we want to detect only highly progressed infections, we add little LuxR. If we want to detect infections with minimum progression, we saturate with AHL.

Thus construct 2 can be used as a sensitivity attenuator.

Packaging

Infector Detector can be packaged as either a cream or a spray.

A spray will provide easy application of the detector because it does not require the user to fiddle around with the urinary catheter as he can simply spray from a distance. The disadvantage being poor accuracy of application and more evaporation.

A cream on the other hand will decrease significanlty any evaporation and will allow the user to apply the detector to specific areas of the catheter without the possibility of spraying the patient itself with detector. The disadvantage being here is the low diffusion rates of AHL and other compounds that need to cross the viscous cream to reach the actual detector assemblies. This might hinder rapid detection.

Both applications provide some advantages and disadvantages that must be weighed depending on the actual use scenario of Infector Detector in order to decide which is best.

<< F2620 Comparison | Conclusions | Home >>

References

1. Damien Balestrino et al. Characterization of Type 2 Quorum Sensing in Klebsiella pneumoniae and Relationship with Biofilm Formation. J Bacteriol. 2005 April; 187(8): 2870–2880.