Tecan Analysis

POUR CELUI QUI MANIP LE TECAN QUAND JE NE SUIS PAS LA

1) Préparer les dilutions de Fer. Comme convenu la semaine dernière, nous ne nous étalerons plus sur els dilutions a 10^-10 ou -9, mais nous nous focaliserons sur 10^-3 à 10^-6 (soit 4 dilutions différentes).

Pour 12 puits de cultures, on aura besoin de 200x12=2400 µL de milieu à la bonne concentration de Fer. Pour 24 puits de cultures, on aura besoin de 4800 µL (suivant le nouveau plan de Tecan où l'on réduit les concentrations).
On fera donc un calcul pour 5000 µL de culture.
Sachant que la concentration de Fer de la solution mère est de 10^-2, on mettra 555µL de cette solution mère dans 5mL de milieu M9 iron free (M9IF), obtenant ainsi une dilution à 10^-3. Ensuite, prélever 555 µL de la solution à 10^-3 et mettre dans les 5000 µL de M9IF du tube devant contenir la dilution à 10^-4, et ainsi de suite pour les dilutions à 10^-5 et 10^-6.

Goal

The Tecan is a plate reader that is able to characterize our bacteria of interest by measuring, for example, growth rate (OD) and/or protein production (GFP). The software saves the measurements over time, thus allowing to study the kinetics of the studied phenomena. With a 96 wells plate (8x12), we are able to test various conditions.

Steps

The hardest part when you use a plate reader to characterize your parts is to carefully choose your controls and anticipate the timings of your bacterial pre-cultures.

We tested various controls during this summer:

• TOP10 growth control
  
• BL21 growth controlFor GFP expression: GFP expression under the control of a pLac promoteLac operator in a delta-LacI strain and GFP expression under the control of a Fur operator in a Δ-Fur strain.

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:

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:

Technical triplicate are made to see the variation induced by the manipulator.
Biological duplicate are made to determine the natural variation of the process observed.

Cycles

AJOUTER DETAIL DES CYCLES

Analysis