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

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Beta-Galactosidase Assay

Note: seeing that the beta-galactosidase assay cannot be used due to size restrictions in vitro, the second possibility - alkaline phosphatase [BBa_J61032] is currently being considered. Also, DsRed Express can also be used.

The gene lacZ encodes for the enzyme ß-galactosidase, which naturally hydrolyzes lactose to glucose and galactose. ß-galactosidase can also hydrolyze lactose homologues to give chromophores. We will be using o-nitrophenyl-ß-galactoside as a substrate for ß-galactosidase, which can be hydrolyzed to a chromophore which absorbs at 420 nm (575 nm for CPRG).

Z-Buffer
  • 60 mM Na2HPO4
  • 40 mM NaH2PO4
  • 10 mM KCl
  • 1 mM MgSO4
  • 50 mM 2-mercaptoethanol
  1. Adjust the pH to 7.0 with 10 M NaOH
  2. Add 4 mg/mL o-nitrophenyl-β-D-Galactoside (ONPG)
  3. Store at 4°C, is it recommended that ONPG is added prior to use to prevent spontaneous hydrolysis
Stop Solution
  • 1 M Na2CO3
Protocol
  1. Cells should be lyzed by specific technique
  2. Incubate cell lysate at 30°C for 10 minutes
  3. Add ~1000 µl Z-buffer with ONPG and note the starting time
  4. Add ~500 µl stop solution and note the ending time
  5. Transfer solution to cuvette and measure absorbance at 420 nm
    • Absorbance should ideally be from 0.6 to 0.9#
    • Cell debris can interfere at 420 nm; cell debris absorbance can be measured at 550 nm
      Centrifuging the sample can reduce cell debris
    • Cell count measurement at 600 nm can also give more accurate calibration
Notes

[Do we use ONPG (Sigma N1127) or the more sensitive CPRG (Sigma 59767)?]
ONPG has a extinction coefficient of 4500 M-1cm-1
 
The protocols for the ß-galactosidase assay can be found in A Better Miller.
Alternatively, another protocol can be found at Rothlab.

  • If high levels of B-galactosidase are being assayed add 0.1ml of culture to 0.9ml of Z buffer
  • If low levels are being determined add 0.5 ml of culture to 0.5ml of Z buffer
  • OD420 = 1.75 x OD550

Experiment 3

Detector system: Chassis: E coli, DNA: pLux with hrp system, Reporter: B-D-Galactosidase
Signaling system: AHL induced

  • Measure of B-D-Galactosidase response to varying AHL concentrations:
    1. Same as exp A
  • Measure of B-D-Galactosidase 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: B-D-Galactosidase
Signaling system: AHL induced

  • Measure of B-D-Galactosidase response to varying AHL concentrations:
  • Measure of B-D-Galactosidase 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: B-D-Galactosidase
Signaling system: AHL induced

  • Measure of B-D-Galactosidase response to varying AHL concentrations:
  • Measure of B-D-Galactosidase response to varying temperature conditions:
  • Measure of life-span of system

Experiment H

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

  • Measure of B-D-Galactosidase response to varying AHL concentrations:
  • Measure of B-D-Galactosidase response to varying temperature conditions:
  • Measure of life-span of system

Experiments with E. coli as a model of biofilm

Experiment I

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

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

Experiments with biofilm

Experiment J

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

  • Test detector system on biofilm produced in lab
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