Internship report

Discovery of the laboratory life

Author: Nicolas SENECAUT

Supervisor: Tamara MILOSEVIC

From 6 to 10 January 2014 Inserm U1001

THANKS:

I would like to thank Tamara and all the people met at the 6th floor who took the timeto explain their projects and help us to understand better the research work.

I am especially grateful to Tamara, Maria and Alice for their welcome and theirkindness.

Discovery of the laboratory's life

Abstract:

During this internship I have discovered a lot of things and participated in a research'sproject.For this I had the opportunity to run many experimentations, this was very interesting.And I met many researchers of the 6th floor who work in many different projects.

Discovery, Laboratory, yeast, microbiology, Antibiotic, spectrophotometry

Résumé:

Durant ce stages j'ai découvert beaucoup des choses et j'ai participé à un projet de recherche.Pour cela j'ai eu l'opportunité fde aire beaucoup de manipulations, cela a été très intéressant.Et j'ai pu rencontrer des chercheurs au 6 éme étage Ils travaillent sur beaucoup de projets.

Découverte, Laboratoire, Levures Projet, Antibiotique

Graphical abstract

Table of content

pages

6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction

7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Context and Activity report

8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . My handling

9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Best shacking

11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Testing geneticin

13 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and supplementary 14 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . About the author

15 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References

Introduction

This internship took was during the week of January 6, 2014 in TaMara's lab. I was inpairs with Valentine Le Gall, another L1 student.

My objective was to discover the laboratory life for my first lab internship. Which allowed me to discover a lot of things and run many experiments. Moreover I met many researchers of the 6th floor who work in many different projects .

what is yeast ? Yeast is unicellular eukaryotic cell whith a “sex” A or α.Yeast can reproduce in 2 differents waysThe first is by division (1)The cell mother budding to form another cell daughter the second is by sporulation (2)Sporulation is formed when the environment is not favorable for the cell of yeast.To protect itself it fuses with another cell, of a different sex, to form a diploid cell who divides in 4 spores 2 A and 2 α.This form is very long-lasting and can stay a very long time without damage. Then, when the media is more favorable to the normal life, spore decomposes its ascus (kind of protective shell which keeps the spore united).

The yeast cell's life cycle:1. Budding

2. Conjugation3. Spore

why are we us ing yeasts ? We use yeasts because they are not expensive and they reproduce themselves quickly (more or less every two hours).Then more specifically, we used budding yeast (Saccharomyces cerevisiae) because its genome was sequenced and the genes are known. Some experiments have studied their mutation.One of the biggest advantages is that yeast cells are eukaryotic cells, like the human

cells, but they don't have the same impact on the ethics (The human is sacred !)And being a unicellular organism there is no inter-cellular interaction that disturbs their studies.

How does it work ? In the lab, Wildtype Yeasts are grown in liquid or solid media called YPD (Yeast Extract Peptone Dextrose).Indeed, this media contains peptone and yeast extract to provide essential amino acids, glucose as an energy source. Under appropriate conditions, yeast can divide every 2 hours.Thus, for example a single colony (about 10^6 cells) dispersed in 10ml of liquid YPD can give 14 times the world's population after an overnight of incubation at 30 °C (10^7-10^8 cells/ml.)Then we can study their growth by using spectrophotometer, which measures opacity density and reveals the yeast ability to divide.For high-throughput experiments; We can use the TECAN which is an automatic spectrophotometer that gives us different values depending on the time.My objective of this internship is to discover the laboratory life and run experiments.

Context/Background

what is the maria's project ?

Maria POTHIER is Tamara's trainee she does her L3 internship in this lab because she loves experimentations and she wants to be in a lab where she is sure she can do her project.The subject of Maria Project is “ Determining the impact of the loss of one gene on the growth of a yeast cell” She wants to find the time taken by a yeast to express the phénotypes by a mutation on her genomes.To do that she had to run a lot of experiments.

WHAT I 'VE D ONE :

Activity report

Our Supervisor TAMARA presented her thesis « the genetics of death on the yeast »I have learned that yeasts have more than 60000 genes and 19 % of them is essential to the growth, approximately 11000 genes.And the more likely cause of death is a genetic problem with the tRNA

Then Maria, her trainee presented us her internship's project.

It was about :“Determining the impact of the loss of one gene on the growth of a yeast cell”After dissection of spores of a mutant yeast, each colony formed is arranged in each well in a 96 wells plate.The next day after an optimal growth of the yeast of each well (seen by the TECAN) we must separate the mutated spores from those which are not.But how ? Maria found a solution: we just had to transfer the plate but this time we added geneticin. Geneticin is an antibiotic who kill euckariotic's cells whitsout neo resistance gene (coding for aminoglycoside phosphotransferase)

To prove her results, she needed to add witnesses (positive control and negative control).Positive control are wildtype yeast (diploid cell) they are resistant to the antibiotic.Negative control are wild type yeast (diploid cell) which have no resistance to this antibiotic.

My Handling :

We had the opportunity to handle a lot during this internship.our objective was to help Maria in her internship's project.Indeed we have to find what is the best concentration for geneticin to have a ideal graph like that:

Then we had to transfer the cells in each well with YPD to re-grow them because they were in another plate in the fridge.Pipetting 96 times was a bit boring but it shows the downs in research's work.

Finally we added oil to avoid the evaporation in the machines.

We have let the plate overnight in the TECAN.we have exploited the excel's table created by the TECAN. It has shown a big difference between the single measure and the multiple measure per well.

BEST SHACKING FOR HAVING A DISPERCED CELLS:

We have looked the plate and saw yeasts were grouped on the center. It was a big problem with the shaking so we have measured the time taken by the TECAN to take its measure. It took more time to measure (15mins) than shaking (10mins) For the day after we had to choose many different ways of shacking.We have tested many different shackings and we have found that it was in decreasing the amplitude to 1mm and choose orbital's shaking that the yeast were more dispersed.

This is the first plate, we can see that the yeast have sedimented

And this is the last plate, we can note that yeast are more dispersed

TESTING THE GENETICIN CONCENTRATION:

Then, we went on with Maria's experience by transferring the plate and adding

geneticin.we have exploited the table, It's shown a big Problem:First we have missed the control and an experiment without witness is not a empiricalprotocol.Secondly a well where nothing should have happened had grown then we assumed contamination.

This plate doesn't have contamination because there are only yeasts.

Only the control have grown, it shows that the concentration of the antibiotics is too high.

For the next day we had to make a plate with different concentrations to see the idealconcentration.And a well which shouldn't have grown had grown so we had to plate it to see the contamination.To be sure, we put the contents of this well on a plate.

This plate shows a contamination because the colonies have 2 colors, which shows two different species.

We have run the same experiment created a 96 well plate with different concentrations of geneticin.We have to calculate the proportions.But to do that we have some problems.First the used YPD was contaminated.Secondly we hadn't made the Yeast solution again in a new YPD the day before so theYeasts were tired.Thirdly we have to prepared a new tube with YPD and Geneticin.

This is Finale table: in yellow is negative control and the rest is positive control and wells with mutated yeasts.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

0,1

1

F5

G5

H5

A5

B5

C5

D5

E5

Conclusion and perspectives

This internship was very cool so this allowed me to meet the people who live in the 6th floor and meet researchers and Phd students.What I learned of this introduction of the work of searcher is:Historic searchers had to search how to get the results in which experiment. And theyalready manipulate in the laboratory with a lab coat. Now researchers have all the data (Big data)and they have to interpret them and search how to model it.They haven't the opportunity(chance) to go to the lab rather than being on the computer and even sometimes they pay someone to manipulate for them.I also learned that in the “profession of researcher” we have to choose our place.Indeed, whether you must be a very popular person who has already published a lot and you can go to CNRS or you are a “normal person” and you have to go in private lab or to teach.Because it is difficult to combine this special work with a family life.We discovered how to shake when the cells are agglutinated.

Supplementary material

We have met a lot people:

we were presented the MOOC's project of Julien Bariére (L2 students), it was very interesting.

Francois-Xavier PELLAY is a private researcher at Dipta (BIODERMA/ESTHED) and a public researcher at the inserm's lab (U1001). So he found the compromise between public and private research.His project is studying the aging of bacteria especially the series of reaction in the mitochondria.He has found molecule which can stop the creation of the ROS (reactive oxygen species).ROS are free radicals made by the incomplete reactions in the mitochondria.To help him we had to run a experiment.We had to create a concentration range of bacteria and different “anti-ROS” molecules found by François-Xavier. We measured the growth rate using an automated spectrophotometer Tecan.

FACS with Chantal LOTTON and Tatiana DIMITRIUTatiana is a PhD Student at the PhD program FDVTatiana's project is to study cooperation between bacteria. FACS (Fluorescence-activated cell sorting) machines are really big machines which count the cells in a volume of liquid and recognize the different cells by color.The lab U1001 doesn't have money to buy a FACS machine (because it costs 200 000euros ! 2 porches) That is why they have to rent a machine in a hospital to run their experimentsBut since the machine is already used for eukaryotic cells (mainly for human cells), they have to “fix” the bacteria, which means that they have to kill the bacteria and forconservation they use glycerol (similar to formaldehyde).It gives a very complex graph representing cell proportions by colors. Since the bacteria were colored by their function, we get statistics based on each proportion.

About the author -

I am in the first year of the bachelor's program “Frontiére du vivant”. I did my high school studies focusing on biology. Thus because I like science I have chosen this bachelor. I love to do many experiments and the lab is for me a sanctuary. This internship was my first lab internship.During this internship I was able to better understand the work of a researcher.Especially the fact that there is less handling but more and more exploiting of large data.

References

“Yeast_mutants_Maria_03.12.2013.pdf” Maria's internship presentation

"http://en.wikipedia.org/wiki/Yeast”Yeast generral definition

“Yeast as a Model Organism ”David Botstein, Steven A. Chervitz, and J. Michael Cherry

Department of Genetics, Stanford University School of Medicine, Stanford, CA94305, USA

“Yeast: An Experimental Organism”for 21st Century Biology

David Botstein*,1 and Gerald R. Fink†*Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton,

New Jersey 08544, and †Whitehead Institute for Biomedical Research

“http://forums.futura-sciences.com/biologie/41108-geneticine-antibiotique.html”action of geneticin