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

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&deg;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&deg;C for 10 minutes
 * 3) Add ~1000 &micro;l Z-buffer with ONPG and note the starting time
 * 4) Add ~500 &micro;l stop solution and note the ending time
 * 5) Transfer solution to cuvette and measure absorbance at 420 nm
 * 6) *Absorbance should ideally be from 0.6 to 0.9#
 * 7) *Cell debris can interfere at 420 nm; cell debris absorbance can be measured at 550 nm Centrifuging the sample can reduce cell debris
 * 8) *Cell count measurement at 600 nm can also give more accurate calibration

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:
 * Same as exp A
 * Measure of B-D-Galactosidase response to varying temperature conditions:
 * Same as exp A
 * Measure of life-span of system
 * Same as exp A

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

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

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

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