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 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 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 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

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: 4 days
• Status: Scheduled for 9th August

Equipment

Day 1:

• 1 eppindorf of competant cell
• 10 µl of DNA
• 0.5 ml LB medium
• 1 ampicillin plate

Day 2:

• 40 ml LB medium
• 40 µl ampicillin stock (50 mg/ml)
• 8 sterile tubes (30 ml)

Day 3:

• 5x 550 ml LB medium in conical flasks
• 2.75 ml ampicillin stock (50 mg/ml)

Day 4:

• Water bath (30°C)
• 21 sterile tubes (30 ml)
• Stop watch/timer
• ??? g 3OC₆HSL (??? mg/ml)
• Fluorimeter

Protocol

Day 1:

1. Transform plasmid into cells using electroporation
2. Grow in 0.5 ml LB for 1 hour
3. Plate on ampicillin plate and leave overnight at 37°C with shaking

Day 2:

1. Make 8 of 5 ml LB medium with 5 µl ampicillin in sterile tubes
2. Pick 8 colonies and innoculate in tubes
3. Leave overnight at 37°C with shaking

Day 3:

1. Make 5 of 550 ml LB medium with 550 µl ampicillin in sterile conical flask
2. Innoculate in 5 cultures into the the conical flasks
3. Leave overnight at 37°C with shaking

Day 4:

1. Prepare a water bath at 30°C [should we use a shaking incubator?]
2. Aliquot the 550 ml cultures into 25 ml sterile tubes
3. Add 53.3075 ng AHL each time to increase [AHL] in steps of 10nM
4. Begin the timer when AHL is added
5. At 5 minute intervals for the first 40 minutes, and 10 minute intervals thereafter:
   1. Sample a 1 ml of the reaction mixture
   2. Immediately chill in ice (to slow the rate of reaction)
   3. Centrifuge at maximum speed for 30 seconds to pellet the cells
   4. Remove the supernatant and resuspend the pellet in 1ml distilled water
   5. Put in a spectrometer and excite it at 501nm and detect at 511nm
      [Note: Use solution at 0 minutes as a blank, assume no fluorescence is present prior to activation]
6. These measurements should be repeated with the other 4 cultures of bacteria

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 robustness of the 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 temperature on the robustness of the system.

Variables and Controls

• Independent variable: Temperature
  • Range: 20 - 40°C
  • Steps/Intervals: 5°C
• Dependent variable: Change 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): 3
• Predicted length of experiment: 4 days
• Status: unscheduled

Equipment

Day 1-3:

• Uses identical equipment to the previous experiment

Day 4:

Protocol

Day 1-3:

• Grow 3 cultures of E. coli transfected with the construct in 550 ml LB medium (as in previous experiment)

Day 4:

1. [We need to find a way to maintain a constant temperature]
2. Aliquot the 550 ml cultures into 25 ml sterile tubes
3. Add ??? ng AHL to each culture and begin the timer
4. At 5 minute intervals for the first 40 minutes, and 10 minute intervals thereafter:
   1. Sample a 1 ml of the reaction mixture and immediately chill in ice
   2. Centrifuge to pellet the cells, and rince them with distilled water
   3. Put in a spectrometer to measure the fluorescence of GFP
5. These measurements should be repeated with the other 2 cultures of bacteria

Test for temperature dependence of the lifespan

Detector system: Chassis: E. coli, DNA: pLux without hrp system, Reporter: GFP
Signaling system: AHL induced
The sensitivity of the system to temperature is to be determined through this experiment. To do this, we induce E. coli cells transfected with the construct with a known concentration of HSL. Different cell cultures are kept at different constant temperatures and their response measured
Aims:

• To determine the rate of change of protein production relative to temperature
• To determine the degradation of the system relative to different temperatures

Variables and Controls

• Independent variable: Temperature
  • Range: 20 - 40°C
  • Steps/Intervals: 5°C
• Dependent variable: Rate of Fluorescence
  • Sampling intervals:
  1. Monitor once daily
• Repetitions of experiment(n): 3
• Predicted length of experiment: 1 - 5 days

(Important Requirement: Experiment should be started on a Monday)

• Status: scheduled for ?? August

Protocol

• 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

Other Experiments

Attached is a list of experiments to be planned.