"
Team:Evry/Biology/RNAseq/ExperimentalDesign
From 2014.igem.org
Cell culture
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:
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.(link)
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.
- cadmium : 300uM
- lead : 100 ppm
- nitrite : 0,9 ng/L
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.(link)
