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

Biofilm Detection: Implementation

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

Protocols

Experiment A

Chassis: E coli, DNA: pLux without hrp system, Reporter: GFP

• Measure of GFP response to varying AHL concentrations:
  1. Renew the bacteria with an 25ml of LB with ampicillin (?)
  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

Chassis: E coli, DNA: pLux without hrp system, Reporter: Luciferase

1. Measure of GFP response to varying AHL concentrations:
2. Measure of GFP response to varying temperature conditions:
3. Measure of life-span of system

Experiment C

Chassis: E coli, DNA: pLux with hrp system, Reporter: Luciferase

1. Measure of GFP response to varying AHL concentrations:
2. Measure of GFP response to varying temperature conditions:
3. Measure of life-span of system

Experiment D

Chassis: In-vitro, DNA: pLux without hrp system, Reporter: GFP

1. Measure of GFP response to varying AHL concentrations:

1. Measure of GFP response to varying temperature conditions:
2. Measure of life-span of system

Experiment E

Chassis: In-vitro, DNA: pLux with hrp system, Reporter: Luciferase

1. Measure of GFP response to varying AHL concentrations:

1. Measure of GFP response to varying temperature conditions:
2. Measure of life-span of system

Experiment F

Chassis: In-veso, DNA: pLux without hrp system, Reporter: GFP

1. Measure of GFP response to varying AHL concentrations:

1. Measure of GFP response to varying temperature conditions:
2. Measure of life-span of system

Experiment G

Chassis: In-veso, DNA: pLux without hrp system, Reporter: Luciferase

1. Measure of GFP response to varying AHL concentrations:

1. Measure of GFP response to varying temperature conditions:
2. Measure of life-span of system

Experiment H

Chassis: In-veso, DNA: pLux with hrp system, Reporter: Luciferase

1. Measure of GFP response to varying AHL concentrations:

1. Measure of GFP response to varying temperature conditions:
2. Measure of life-span of system

Biobricks

• Construct, clone and growth over 2 day period