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

Biofilm Detection: Implementation

• Introduction
• Specifications
• Design
• Modelling
• Implementation
• Testing/Validation
• Notes
• References

Biobricks

• Construct, clone and growth over 2 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.

Testing for AHL Sensitivity

Overview

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 increase 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 increase 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

Variables and Controls

• Independent variable: [AHL]
  • Range: 0 - 200nM
  • Steps/Intervals: 10nM
• Dependent variable: Increase in GFP fluorescence over time
  • Response: Over period of 2 hours
  • Sampling intervals:
    1. 5min over first 40min
    2. 10min thereafter
• Repetitions of experiment(n): 5
• Predicted length of experiment: 2 days
• Status: Scheduled for 9th August

Protocol

• Measure of GFP response to varying AHL concentrations:
  1. Renew the bacteria with an 25ml of LB with 312.5 µl ampicillin (4 mg/ml) [?]
  2. Add 53.3075ng AHL each time to increase [AHL] in steps of 10nM
  3. Keep in a water bath at 30°C
  4. Take a 1ml sample of the solution and put it in ice (to slow the rate of reaction)
  5. Take samples every 5 minutes for the first 40 minutes and every 10 minutes thereafter
  6. Centrifuge at maximum speed for 30 seconds
  7. Remove the supernatant
  8. Resuspend the pellet in 1ml distilled water (ensure the entire pellet is resuspended)
  9. Put in a spectrometer and excite it at 501nm and detect at 511nm

[Use solution at 0 minutes as a blank]

• Measure of GFP response to varying temperature conditions:
  1. Renew the bacteria with an 25ml of LB with ampicillin (?)
  2. Add ??? ng AHL to the reaction mixture
  3. Keep reaction mixtures at different temperatures from 20°C to 40°C
  4. Take 1ml samples and record fluorescence as above

• Measure of life-span of system
  1. Grow the bacteria in 15ml of LB with ampicillin
  2. Keep reaction mixtures at different temperatures from 20°C to 40°C
  3. Add ??? ng AHL to the reaction mixture
  4. Take 1ml samples and record fluorescence as above

Experiment B

Detector system: Chassis: E coli, DNA: pLux without hrp system, Reporter: Luciferase
Signaling system: AHL induced

• Measure of GFP response to varying AHL concentrations:
  1. Same as exp A
• Measure of GFP response to varying temperature conditions:
  1. Same as exp A
• Measure of life-span of system
  1. Same as exp A

Experiment C

Detector system: Chassis: E coli, DNA: pLux with hrp system, Reporter: Luciferase
Signaling system: AHL induced

• Measure of GFP response to varying AHL concentrations:
  1. Same as exp A
• Measure of GFP response to varying temperature conditions:
  1. Same as exp A
• Measure of life-span of system
  1. Same as exp A

Experiments in Vitro

Experiment D

Detector system:Chassis: In-vitro, DNA: pLux without hrp system, Reporter: GFP
Signaling system: AHL induced

• Measure of GFP response to varying AHL concentrations:
• Measure of GFP response to varying temperature conditions:
• Measure of life-span of system

Experiment E

Detector system:Chassis: In-vitro, DNA: pLux with hrp system, Reporter: Luciferase
Signaling system: AHL induced

• Measure of GFP response to varying AHL concentrations:
• Measure of GFP response to varying temperature conditions:
• Measure of life-span of system

Experiments in veso

Experiment F

Detector system:Chassis: In-veso, DNA: pLux without hrp system, Reporter: GFP
Signaling system: AHL induced

• Measure of GFP response to varying AHL concentrations:
• Measure of GFP response to varying temperature conditions:
• Measure of life-span of system

Experiment G

Detector system:Chassis: In-veso, DNA: pLux without hrp system, Reporter: Luciferase
Signaling system: AHL induced

• Measure of GFP response to varying AHL concentrations:
• Measure of GFP response to varying temperature conditions:
• Measure of life-span of system

Experiment H

Detector system:Chassis: In-veso, DNA: pLux with hrp system, Reporter: Luciferase
Signaling system: AHL induced

• Measure of GFP response to varying AHL concentrations:
• Measure of GFP response to varying temperature conditions:
• Measure of life-span of system

Experiment I

Detector system:Chassis: In-veso, DNA: pLux with hrp system, Reporter: Luciferase
Signaling system: AHL secreting Bacteria

• Test detector system with AHL secreting E coli, as a model of the biofilm

Experiment J

Detector system:Chassis: In-veso, DNA: pLux with hrp system, Reporter: Luciferase
Signaling system: Biofilm produced in lab

• Test detector system on biofilm produced in lab