Team:Evry/Biology/RNAseq/ExperimentalDesign

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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 (lien). So, we have tested 3 compounds on marine broth.

nitritecadmium
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.
lead stress nitrite stress cadmium stress
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
because that modified the bacterial growth but do not kil it. So, we must to be check the quality of RNA. 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 extraction from Tolonen Lab. protocol of RNA Throughout of the experiment, electrophoresis gels are done in order to confirm which have always RNA. Gel1Gel2 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. result1result2 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. Gel3Gel4 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.(link)

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