Team:Paris Bettencourt/Experiments/Microscopy


Team IGEM Paris 2011



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 different type of strains in order to mix them later for the nanotube observation or use them separately for a positive or negative control which will confirm or not our assumptions.
  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 a certain optical density. The optical density we need to reach for E.coli is 0.4 and 0.8 for Subtilis.
  3. We centrifuge the cultures for 10' at a velocity of 4000 rpm. Afterward we throw the supernatant LB and we concentrate the cultures into 150 micro liters of LB, allowing us to have good conditions of observation. Indeed, we need the cells to be aglutinated into a dense monolayer.
  4. We pipette one of the strains onto one of the wells, which will constitute our positive/negative 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.

Detailed GFP diffusion experiments

Experiments with Ben-Yehuda's strains

The first GFP diffusion experiments were done with Ben-Yehuda's strains: PY79 (non fluorescent strain) and PY79+S12 (fluorescent strain). These experiments allowed us to train on the slide preparation and do precise adjustments on the microscope. As the intensity of the fluorescence was not high enough in order to allow us to make reliable observations, we did not use these strains for a long time. You can find the details of the experiments on the notebook.

Experiments with wild-type strains

First experiment with wild-type strains

We decided to use a wild-type strain 3610 (non fluorescent strain) and 3610 GFP. With this strain we obtained better microscopy results (pictures and videos) since the fluorescence was stronger than with the PY79 strain. Hence we decided to continue our GFP diffusion experiments with the 3610 strain. You can find the details of the first experiment with this strain on the notebook.

First successful experiment with wild-type strains

We obtained our first satisfying results with the wild-type strains. Indeed one of the experiments which was done with the 3610 strain allowed us to clearly observe GFP diffusion and thus consitutes one of our main demonstrative experiments. You can find the details of this experiment on the notebook and on the GFP diffusion experiment page.