Team:Paris Bettencourt/Experiments/Microscopy

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Team IGEM Paris 2011

Microscopy

Overview

The main goal of the microscopy experiments is to obtain pictures and movies that constitutes a reliable proof of concept for our different genetic systems. Our first experiment consisted in reproducing Ben-Yehuda's results on GFP diffusion. The results of this important demonstrative experiment can be found here

    Preparation of the slide

    Fig 1: Global view of the material used for the preparation

    In all the microscopy experiments that will later be described we will use a two-well microscopic slide; one dedicated to a control and another one that will contain the actual experimented mix. Here is the description of its preparation:

    1) We melt sterilized Luria Broth with 1.5% of Agarose ("LBA") by micro waving it.

    2) We dispose two drops (for each well) 160 micro liters of LBA on a lamella on which we indicated a center reference.

    Fig 2: Lamella with the center references in red

    3) We superimpose the excavated part of the slide on the center reference of the LBA.

    4) We let the preparation cool down in a fridge for ten minutes in order to allow the LBA to dry.

    Fig 3: Preparation is left to dry in the fridge

    5) We take out the preparation and carefully withdraw the lamella from the slide.

    6) We cut up the LBA with a scalpel such as getting 3 millimeter width rectangles parallel to the length of the slide.

    7) We pipette a 1 micro liter of the culture on the LBA and let it dry for few minutes.

    Fig 4: The culture is pipetted on the LBA rectangles

    8) We lay down two squared lamella on each well.

    9) We dispose nail polish on the lamella's corners to stick it to the slide.

    10) We dispose hydrophobic grease on the edges of the lamellas in order to avoid a premature dry of the cultures.

    Fig 5: Slide is ready for the microscope

    Culture preparation protocol

    Let's describe the principal steps of the protocol we used:

    1) We put into overnight two different type of strains: one fluorescent strain and one non fluorescent strain.

    2) The following morning we dilute the cultures in order to reach an optical density of 0.1, then we let them grow up until reaching an optical density of 0.8.

    3) We centrifuge them and concentrate the cultures into 150 micro liters of LB, allowing us to have good conditions of observation.

    4) We pipette the fluorescent culture onto one of the wells, which will constitute our positive control; then we pipette a mix of both strains onto the second well. (see the upside protocol describing the preparation of the slide).

    5) We make observations by using a phasecontrast and fluorescence microscope with a 100 times magnification. The acquisition system is controlled by MetaMorph. The expected phenomenon is the illumnation of some cells that are next to fluorescent cells.

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