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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 hours	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 type of 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. By using a construct that recognises AI-2¹, for example, 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.

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 they can simply spray from a distance. The disadvantage is the poor accuracy of application, waste, and higher rate of evaporation.

A cream, on the other hand, will decrease significantly any evaporation and will allow the user to apply the detector to specific areas of the catheter with more control. The disadvantage is that the diffusion rate of AHL and the detection compounds through a viscous cream is lower. This will slow down the response of the system.

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.

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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.