IGEM:IMPERIAL/2007/Projects/Biofilm Detector/Implementation

=Infector Detector: Implementation=

 Introduction Specifications Design Modelling Fabrication Testing Data Analysis Validation Notes</li> References</li> </ul> <br style="clear:both">

Biobricks

 * Construct, clone and growth over 5(??) day period
 * General protocols for cloning and making the constructs: General Protocols
 * Constructs to be made:Design

Experiments in E. coli
Our initial aim is to test our simple systems in the E. coli chassis. E.coli is a well defined chassis in which most system function. This first phase of experimentation is to ensure that our system works.

Test for AHL Sensitivity
Detector system: Chassis: E. coli, DNA: pLux without hrp system, Reporter: GFP Signaling system: AHL induced The sensitivity of the system is to be determined through this experiment. To do this, we induce E. coli cells transfected with the construct with known concentrations of HSL. We then record the change in GFP, such that we can calculate the rate of GFP production relative to concentration of AHL in solution. Aims:
 * To determine the rate of change of protein production relative to concentration of AHL
 * To determine the response time relative to concentration of AHL
 * To determine the steady state response of protein relative to concentration of AHL

Test for temperature Sensitivity
Detector system: Chassis: E. coli, DNA: pLux without hrp system, Reporter: GFP Signaling system: AHL induced We then need to determine the response of system at different temperatures, allowing us to find out the optimal temperature for the system. This experiment will be similar to the previous one, except with a varying temperature, while maintaining a constant AHL level. The AHL level chosen will be ideally at the concentrations of AHL which the lux promoter is most sensitive to. Aims:
 * To determine the general effect of temperature on the system and determine the optimal temperature

Test for temperature dependence of the lifespan
Detector system: Chassis: E. coli, DNA: pLux without hrp system, Reporter: GFP Signaling system: AHL induced The system must be stable for long periods of time before use. Temperature would be one of the leading factors limiting the shelf life of the system. We therefore want to determine the effect of temperature on the lifespan of the system. The system will be left at a constant temperature for several days. The decline in response time and strength over the days will be measured, through which we hope to determine the shelf-life and optimal storage temperature of the system. Aims:
 * To determine effects of temperature on the robustness of the system (response time and strength)

Test for AHL sensitivity
Detector system: Chassis: E coli, DNA: pLux without hrp system, Reporter: B-D-Galactosidase Signaling system: AHL induced This experiment will be carried out to compare the sensitivity of the system with X-Gal to that with GFP. To do this, we induce E. coli cells transfected with the construct with known concentrations of HSL. We then record the change in X-GAl, such that we can calculate the rate of X-gal production relative to concentration of AHL in solution.

Test for temperature Sensitivity
Detector system: Chassis: E. coli, DNA: pLux without hrp system, Reporter: B-D-Galactosidase Signaling system: AHL induced This experiment will determine the response of system at different temperatures, allowing us to find out the optimal temperature for the system. The value can then be compared to that got through the system with GFP as the reporter.

Test for temperature dependence of the lifespan
Detector system: Chassis: E. coli, DNA: pLux without hrp system, Reporter: B-D_Galactosidase Signaling system: AHL induced The experiment will determine the shelf life of the detector system with X-Gal. This value can then be compared to the shelf life of the system with GFP reporter

Other Experiments
Attached is a list of experiments to be planned.