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

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Infector Detector: Implementation


Contents


Biobricks

  • Construct, clone and growth over 5(??) day period
  • General protocols for cloning and making the constructs: General Protocols

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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.

Experiment 1

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
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 sterile tubes, 25 ml per tube
  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 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
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:

  • 1 eppindorf of competant cell
  • 10 µl of DNA
  • 1 ampicillin plate
  • 4 sterile tubes (30 ml)
  • 20 ml LB medium
  • 3x 125ml LB medium in conical flasks
  • 0.375 ml ampicillin stock (50 mg/ml)

Day 4:

  • [Water bath or incubators (30°C)]
  • 25 sterile tubes (30 ml)
  • Stop watch/timer
  • ??? g 3OC6HSL (??? mg/ml)
  • Fluorimeter
Protocol

Day 1-3:

  • Grow 3 cultures of E. coli transfected with the construct in 125 ml LB medium (as in previous experiment)

Day 4:

  1. [We need to find a way to maintain a constant temperature]
  2. Aliquot the 125 ml cultures into sterile tubes, 25 ml per tube
  3. Incubate the culture in their respective temperatures for 10 minutes
  4. Add ??? ng AHL to each culture and begin the timer
  5. 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 rinse them with distilled water
    3. Put in a spectrometer to measure the fluorescence of GFP
  6. 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 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)
Variables and Controls

  • Independent variable: Temperature
    • Range: 20 - 40°C (Might use 37°C , 4°C , -20°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
  • Status: unscheduled
Equipment

Day 1-3:

  • 1 eppindorf of competant cell
  • 10 µl of DNA
  • 1 ampicillin plate
  • 15 sterile tubes (30 ml)
  • 225 ml LB medium
  • 0.225 ml ampicillin stock (50 mg/ml)

Day 4:

Protocol

Day 1-3:

  • Grow 15 cultures of E. coli transfected with the construct in 15 ml LB medium (as in previous experiment)

Day 4-9:

  1. Transfer 1 ml from each culture into an eppendorf
  2. Warm the culture in the eppendorf to 37°C in an incubator for 10 minutes
  3. Add ??? ng AHL to each culture and begin the timer
  4. Leave the reaction for 30 minutes
  5. Centrifuge to pellet the cells, and rince them with distilled water
  6. Put in a spectrometer to measure the fluorescence of GFP

Experiment 2

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.

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