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

Beta-Galactosidase Assay

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.

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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