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 ??? µl Z-buffer with ONPG and note the starting time
4. Add ??? µl stop solution and note the ending time
5. Transfer solution to cuvette and measure absorbance at 420 nm

Notes

[Do we use ONPG (Sigma N1127) or the more sensitive CPRG (Sigma 59767)?
The protocols for the ß-galactosidase assay can be found in A Better Miller.

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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