Tecan Analysis

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 different controls:

• A control for growth (one for each different bacterial strain)
  
• Two controls for 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