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Team:Evry/Protocols/06
From 2013.igem.org
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<h2> Principle </h2> | <h2> Principle </h2> | ||
| - | Tecan is used to characterized bacteria strain: bacteria growth, protein production, etc. The software measure the Optical Density (OD) or the fluorescence of a compound of interest (e.g. a protein fused with GFP) regurlarly, thus allowing to obtain the cinetic of phenomenas. With a 96 (8x12) | + | Tecan is used to characterized bacteria strain: bacteria growth, protein production, etc. The software measure the Optical Density (OD) or the fluorescence of a compound of interest (e.g. a protein fused with GFP) regurlarly, thus allowing to obtain the cinetic of phenomenas. With a 96 (8x12) wells plate, different conditions could be test. |
<h2> Preparation </h2> | <h2> Preparation </h2> | ||
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<h3>Pre-culture preparation</h3> | <h3>Pre-culture preparation</h3> | ||
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In a 15 mL tube, add 2 mL of M9 medium and inoculate BL21 cells (expression strain) from glycerol<br/> | In a 15 mL tube, add 2 mL of M9 medium and inoculate BL21 cells (expression strain) from glycerol<br/> | ||
| - | <br/><br/></p> | + | To inhibit the expression of sfGPF, use M9 with iron, instead of classical M9. |
| + | <br/> | ||
| + | After one night of culture, refresh the precultures by diluting them 200 times in M9 medium (with iron and carbenicillin). | ||
| + | After 8 hours of culture, prepare your wells plate according to the following scheme:<br/></p> | ||
<h2> Analysis </h2> | <h2> Analysis </h2> | ||
Revision as of 09:15, 26 August 2013
Tecan Analysis
Principle
Tecan is used to characterized bacteria strain: bacteria growth, protein production, etc. The software measure the Optical Density (OD) or the fluorescence of a compound of interest (e.g. a protein fused with GFP) regurlarly, thus allowing to obtain the cinetic of phenomenas. With a 96 (8x12) wells plate, different conditions could be test.Preparation
Medium preparation
LB medium emit a side signal. As turbidity (OD) and fluorescence of our sample are measured, M9 medium is use instead.
Composition for 50 mL of:
| Reagent | M9 medium (without iron) | M9 medium (with iron) |
|---|---|---|
| CaCl2 (1M) | 5 µL | |
| MgSO4 (1M) | 100 µL | |
| Glycerol (50%) | 10 mL | |
| Thiamine | 5 µL | |
| NaOH (pH 7.4) | 12.5 µL | |
| H2O | 40 mL | 39 mL |
| FeSO4 (10mM) | - | 50 µL |
| Casamino acids (0.2%) | - | 1 mL |
Once the mixture is prepared, the medium must be filtered to be sterilised using 0.22 µm filter.
Pre-culture preparation
In a 15 mL tube, add 2 mL of M9 medium and inoculate BL21 cells (expression strain) from glycerol
To inhibit the expression of sfGPF, use M9 with iron, instead of classical M9.
After one night of culture, refresh the precultures by diluting them 200 times in M9 medium (with iron and carbenicillin).
After 8 hours of culture, prepare your wells plate according to the following scheme:
Analysis
TECAN analysis
Using the TECAN analysis, we are measuring the capacity of repression of the natural binding site that we extract from the E.coli's genome.
Preculture with M9 medium (with carbenicillin) have been launched for BL21 transformed with our 1st construction:
- Natural Fur Binding Site of Fec A + sfGFP (clone 1, 2, 3)
- Natural Fur Binding Site of Fep A + sfGFP (clone 1, 2, 3)
- Natural Fur Binding Site of Ace B + sfGFP (clone 1, 2, 3)
Note: Preculture have been made in M9 medium with iron in oder to inhibit the expression of sfGFP.
After one night of culture, time the precultures have been refreshed by diluting them 200 times in M9 medium (with iron and carbenicillin).
After 8 hours of culture, the 96 wells plate has been prepare (see following scheme).
AJOUTER DETAIL DES CYCLES
