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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	To Be Determined - Using Stronger fluorescent protein such as DsRed express
Response Time	System needs to have a response time under 3 hours	Systems responds <30minutes and reaches peak fluorescence at 300minutes
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 and Application	System must be portable and convenient to use	To Be Determined- Using our chassis in a spray

Infector detector utilises synthetic biology to fight one of the most common infections rampaging through hospitals worldwide. Urinary catheter infections and catheter-related bacteria in general have been troubling both doctors and patients for years now on since the infection is usually very difficult to detect at an early stage. Infector detector battles the infection with its own weapons by utilising its quorum sensing mechanism to initiate a fluorescence reporter, signaling the presence of a biofilm in the making. Upon noticing the change in colour, medical staff will replace the catheter before the infection spreads. Thus doctors can rest assured that their patients get the protection they need. And all this without invoking a single bacterium, thanks to our Cell-free chassis.

Its evolution cannot however stop here ! While charactrising infector detector we came across certain aspects that can be further developed to enhance its functions. These are described in more detail further below:

Battle a spectrum of infections

The great potential of Infector Detector lies in that it isn't limited to just one type of infection. Adding sensitivity to AHL originating from biofilms is just the beginning. By using different homologues to the LuxR quorum sensing, Infector Detector can be used to battle a range of catheter-related bacterium. 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 advantages of using construct 2 is that it provides an additional control mechanism for our detector meaning that you can tweak the detector sensitivity. In addition by adding purified LuxR the chassis does not have to produce LuxR and so has more energy to produce GFP.
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 detection compounds through a viscous cream is lower. This will slow down system response.

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.

So as you can see, the development of this application can easily be extended into many areas, and even though it is far from being commercially available, it has proven that even the tiniest bits of the microcosm around us can be used to tackle a worldwide problem.

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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.