Imperial/Infector Detector/Design


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== Design Overview ==
== Design Overview ==
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== DNA Constructs ==
== DNA Constructs ==

Revision as of 00:04, 23 October 2007

Infector Detector: Design

Design Overview

IC07 design ID.png

Property Value Design Solution System Level
Health Regulations System Must not be living replicating bacteria Use a Cell Free System e.g. Promega's S30 Cell Extract Chassis
Lifespan System must have a shelf life of 7 days Protease Inhibitor of Cell Extract should ensure degradation of Visual Reporter is Minimal
Proper Packaging should ensure that evaporation of Cell Free system is so low that system can surive for 7 days
Inputs System must be sensitive to AHL concentration between 5-50nM Sensitive AHL receiver to detect low AHL levels Construct
Outputs System must give a visual signal if bacteria is present Couple AHL to a reporter system expressing fluoresent protein eg. RFP Construct
Operating Conditions System must operate within temperature 20-30°C Gene expression systems and protein must be thermostable Construct
System must operate within pH range of 6-8 Gene expression systems and protein must be pH resistant Construct
Response Time System needs to have a response time under 1 hour To be Determined - this is hard to design for Both

DNA Constructs

Construct Design Availability
Imperial ID Design ID1.png
This part can be found as BBa_T9002 in the registry.
Imperial ID Design ID2.png
This part can be found as BBa_J37032 in the registry.

Our first aim is to create a system that can accurately detect the concentration of AHL in solution. We needed a well characterized detector system sensitive to 3OC6HSL which we will attach to a standard GFP reporter to determine its response to GFP. The first construct is appealing as the AHL receiver in front is well-documented as part BBa_F2620, thus giving more assurance in the reproducible nature of our results.

However, the weakness of construct 1 lies in the fact that it does not respond uniformly to constant AHL concentration since the activator protein LuxR is produced by a promoter and not maintained at a constant level. This makes it hard to relate the output of the system to the AHL input. We thought that if purified LuxR could be controlled and added to the system instead (simplify the construct by removing the constitutive LuxR production), this would give us more controllability on the input, thus better reproducibility.

Chassis Selection

Commercial S30 E. coli Cell Extract in bulk solution + packaging to last 7 days

We have chosen to use the commercially available S30 E. coli cell extract made by Promega. After having looking into a variety of different cell-free chassis, we feel that this chassis best suits our needs. In particular this chassis allows us to meet our base requirement of complying with the Health and Safety regulations of the field we are working in, as we do not want replicative bacteria that could potentially be pathogenic to come in contact with urinary catheters.

In addition to complying with health regulations the S30 cell extract is commercially available meaning that it has been shown to work. This is very important for us as it allows our focus to be on tuning the chassis to suit our needs rather than making the chassis work in the first place.