IGEM:IMPERIAL/2007/Projects/Biofilm Detector/Implementation/Under Construction: Difference between revisions
Ben Yi Tew (talk | contribs) mNo edit summary |
Ben Yi Tew (talk | contribs) mNo edit summary |
||
| Line 1: | Line 1: | ||
==Beta-Galactosidase Assay== | ==Beta-Galactosidase Assay== | ||
We will be using o-nitrophenyl-ß-galactoside as a substrate for ß- | 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 | =====Z-Buffer===== | ||
*60 mM Na<sub>2</sub>HPO<sub>4</sub> | *60 mM Na<sub>2</sub>HPO<sub>4</sub> | ||
*40 mM NaH<sub>2</sub>PO<sub>4</sub> | *40 mM NaH<sub>2</sub>PO<sub>4</sub> | ||
| Line 8: | Line 8: | ||
*1 mM MgSO<sub>4</sub> | *1 mM MgSO<sub>4</sub> | ||
*50 mM 2-mercaptoethanol | *50 mM 2-mercaptoethanol | ||
#Adjust the pH to 7.0 with 10 M NaOH | |||
#Add 4 mg/mL o-nitrophenyl-β-D-Galactoside (ONPG) | |||
Stop Solution | =====Stop Solution===== | ||
*1 M Na<sub>2</sub>CO<sub>3</sub> | *1 M Na<sub>2</sub>CO<sub>3</sub> | ||
=====Notes===== | |||
[Do we use ONPG (Sigma N1127) or the more sensitive CPRG (Sigma 59767)?<br> | |||
The protocols for the ß-galactosidase assay can be found in [[Beta-Galactosidase_Assay_(A_better_Miller)|A Better Miller]]. | |||
---- | ---- | ||
Revision as of 08:07, 3 August 2007
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
- Adjust the pH to 7.0 with 10 M NaOH
- Add 4 mg/mL o-nitrophenyl-β-D-Galactoside (ONPG)
Stop Solution
- 1 M Na2CO3
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.
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
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
