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Team:Evry/Biology/RNAseq/ExperimentalDesign
From 2014.igem.org
Experimental design
Why RNA-seq
RNA-seq is a powerful technique to determine the expression of transcripts in specific conditions. It allows to know the relative expression of each gene for an organism. In principle, more RNA are present more protein will be produced. Using this technology is a chance and we wanted to compare the gene expression of Pseudovibrio denitrificans depending on different conditions. These conditions are: M9 media as a reference, or M9 supplemented with cadmium, nitrite, or lead. By comparing the gene expression betwwen conditions and the reference we should be able to find specific highly overexpressed gene.
Mapping these reads to the genome (thanks to the sequencing of Pseudovibrio denitrificans previously done by the team), and applying an expert eye to these results we should be able to find new sensors. These sensors would be created by taking 500 bp upstream these overexpressed genes. These new promoters would be characterized adding a reporting gene as GFP to quantify the expression depending on different concentration of the compounds used for the RNA-seq. RNA-seq is a critical technique because of the RNA-seq stability. Also each sample preparation must be done in same conditions to be properly compared.
Our conditons of pseudovibrio culture was measured by tecan (Infinite M200).
The compounds which have been tested are nitrite, cadmium and lead.
The kinetic parameters were determinated during the characterization of the bacteria characterization of the bacteria.
So, we have tested 3 compounds on marine broth.
Title: Kinetic of pseudovibrio on Marine Broth (MB) with lead, nitrite or cadmium. We have 2 dilutions of the bacteria : 1/5 and 1/10.
The data of the experiments are not clear because the marine broth is a medium which is trouble, that disrupt the measurement of OD.
As we have a problem with the media for the measurement, we change against the M9 medium with casamino acids because this is a clear medium.
In order to look a modification in the bacterial growth, we have to cause a stress in the bacteria with the interest compounds.
Title: Stress of pseudovibrio denitrificans with differents concentrations of compounds.
The compounds were added after 2 hours. After analysis of the data, the concentrations which will be test for the RNAseq are:
- cadmium : 300uM
- lead : 100 ppm
- nitrite : 0,9 ng/L
With the aid of Tolonen Lab, we make the extraction of DNA and RNA.
The DNA of bacteria was sent after extraction for the DNAseq and the results were analyzed and assembled. DNAseq
For to check the quality of RNA, we extract RNA from new culture in log phase with the protocol of RNA protocol of RNA extraction from Tolonen Lab.
Throughout of the experiment, electrophoresis gels are done in order to confirm which have always RNA.
The DNA of bacteria was sent after extraction for the DNAseq and the results were analyzed and assembled. DNAseq
For to check the quality of RNA, we extract RNA from new culture in log phase with the protocol of RNA protocol of RNA extraction from Tolonen Lab.
Throughout of the experiment, electrophoresis gels are done in order to confirm which have always RNA.
Title : RNA controls by electrophoresis gel.
Gel1 : 1.Ladder, 2. Positive control, 3. Pseudovibrio d. and 4.Other bacteria
Gel2 : 1.Ladder, 2. Pseudovibrio d. and 3.Positive control.
And we were sent samples for to drop off on Agilent chip.
This is the results of Agilent chip.
For to know if our RNA is not degrade, we look rRNA ratio ( it have to included between 1 and 2) and the RIN ( included between 7 and 10). This is approved the method of extraction.
If the quality is confirmed, we do again extractions with the culture cells which are subjected a stress with the differents compounds.
A control by electrophoresis gel is done.
Title : Control of RNA presence
Gel1 and 2 : 1.Ladder, 2.Positive Control, 3.Pseudovibrio d., 4.Pseudovibrio d. make stressed by cadmium, 5. Pseudovibrio d. make stressed by nitrite and 5. Pseudovibrio d. make stressed by lead.
The RNA of bacteria was sent and the obtained results were analysed.
