Team:Evry/Biology/RNAseq/ExperimentalDesign

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<h3>Why RNA-seq</h3><br/>
<h3>Why RNA-seq</h3><br/>
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<p>
 
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, the more RNA molecules are present, the more protein will be produced. Using this technology is a chance and we wanted to compare gene expression of <i>Pseudovibrio denitrificans</i> depending on different conditions. These conditions are: M9 media as a reference, or M9 media supplemented with either cadmium, nitrite, or lead. By comparing the gene expressions between conditions and the reference we should be able to find specific highly overexpressed gene. <br/>By mapping these reads to the genome (thanks to the sequencing of <i> Pseudovibrio denitrificans</i> previously done by the team), and applying an expert eye to these results, we could find promoters that can be used to build new biological sensors. These sensors would be created by taking 500 bp upstream these overexpressed genes. The promoters would be characterized adding a reporting gene as the GFP to quantify the expression depending on different concentrations of the compounds used for the RNA-seq. RNA-seq is a critical technique because of the RNA stability. Also, each sample preparation must be done in the exact same conditions than the others to be properly compared. <br/><br/>
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, the more RNA molecules are present, the more protein will be produced. Using this technology is a chance and we wanted to compare gene expression of <i>Pseudovibrio denitrificans</i> depending on different conditions. These conditions are: M9 media as a reference, or M9 media supplemented with either cadmium, nitrite, or lead. By comparing the gene expressions between conditions and the reference we should be able to find specific highly overexpressed gene. <br/>By mapping these reads to the genome (thanks to the sequencing of <i> Pseudovibrio denitrificans</i> previously done by the team), and applying an expert eye to these results, we could find promoters that can be used to build new biological sensors. These sensors would be created by taking 500 bp upstream these overexpressed genes. The promoters would be characterized adding a reporting gene as the GFP to quantify the expression depending on different concentrations of the compounds used for the RNA-seq. RNA-seq is a critical technique because of the RNA stability. Also, each sample preparation must be done in the exact same conditions than the others to be properly compared. <br/><br/>
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Pseudovibrio growth and Our conditions of pseudovibrio culture was measured by tecan (Infinite M200).<br/>
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Pseudovibrio cultures growth in the different conditions were measured by using a Tecan (Infinite M200).<br/>
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The compounds which have been tested are nitrite, cadmium and lead. <br/>
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The compounds we tested were nitrite, cadmium and lead. <br/>
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The kinetic parameters were determinated during the characterization of the bacteria <a href="https://2014.igem.org/wiki/index.php?title=Team:Evry/Biology/CellCharacterization/Culture">characterization of the bacteria</a>.<br/>
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The kinetic parameters were determined during the characterization of the bacterium <a href="https://2014.igem.org/wiki/index.php?title=Team:Evry/Biology/CellCharacterization/Culture">characterization of the bacteria</a>.<br/>
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So, we have tested 3 compounds on marine broth.<br/>
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We tested three compounds in marine broth.<br/>
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<b>
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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.</b> <br/>
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Title: Kinetic of pseudovibrio in Marine Broth (MB) with either lead, nitrite or cadmium. We diluted the bacteria cultures by 1/5 and 1/10.</b> <br/>
<br/>
<br/>
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<p>
 
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The data of the experiments are not clear because the marine broth is a medium which is trouble, that disrupt the measurement of OD.<br/>
 
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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. <br/>
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Because of the marine broth, which is a cloudy rich medium, we were not able to draw conclusions as it prevented to have a correct measurement of the OD.<br/>
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In order to look a modification in the bacterial growth, we have to cause a stress in the bacteria with the interest compounds.
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Due to that problem with the medium, we preferred to use a M9 medium with casamino acids, because it is a clear media. <br/>
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In order to be able to observe differences in the bacterial growth, we had to cause a stress to the bacteria by adding the compounds of interest.
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<br/>
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<br/></p>
 
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The compounds were added after 2 hours.
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The compounds were added after two hours.
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After analysis of the data, the concentrations which will be test for the RNAseq are:
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After analysis of the data, the concentrations we decided to test for the RNA-seq were:
<ul>
<ul>
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<li> cadmium : 300uM
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<li> cadmium : 300uM
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<li> lead : 100 ppm  
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<li> lead : 100 ppm  
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<li> nitrite : 0,9 ng/L
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<li> nitrite : 0,9 ng/L
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</ul>
</ul>
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because that modified the bacterial growth but do not kil it.
because that modified the bacterial growth but do not kil it.
   
   
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Throughout of the experiment, electrophoresis gels are done in order to confirm which have always RNA. <br/>
Throughout of the experiment, electrophoresis gels are done in order to confirm which have always RNA. <br/>
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<br/></p>
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<b> Title : RNA controls by electrophoresis gel. </b><br/>
<b> Title : RNA controls by electrophoresis gel. </b><br/>
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<p>
 
Gel1 : 1.Ladder, 2. Positive control, 3. Pseudovibrio d. and 4.Other bacteria<br/>
Gel1 : 1.Ladder, 2. Positive control, 3. Pseudovibrio d. and 4.Other bacteria<br/>
Gel2 : 1.Ladder, 2. Pseudovibrio d. and  3.Positive control.<br/>
Gel2 : 1.Ladder, 2. Pseudovibrio d. and  3.Positive control.<br/>
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If the quality is confirmed, we do again extractions with the culture cells which are subjected a stress with the differents compounds.  
If the quality is confirmed, we do again extractions with the culture cells which are subjected a stress with the differents compounds.  
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A control by electrophoresis gel is done.</p>
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A control by electrophoresis gel is done.
<img src="https://static.igem.org/mediawiki/2014/4/49/ExtractionPseudovibrio_gel_1.jpg" alt="Gel3" />
<img src="https://static.igem.org/mediawiki/2014/4/49/ExtractionPseudovibrio_gel_1.jpg" alt="Gel3" />
<img src="https://static.igem.org/mediawiki/2014/4/4c/ExtractionPseudovibrio_gel_2.jpg" alt="Gel4" />
<img src="https://static.igem.org/mediawiki/2014/4/4c/ExtractionPseudovibrio_gel_2.jpg" alt="Gel4" />
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<p>
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<b> Title : Control of RNA presence </b>
<b> Title : Control of RNA presence </b>
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.
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.
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The RNA of bacteria was sent and the obtained results were analysed.<br/></p>
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The RNA of bacteria was sent and the obtained results were analysed.<br/>
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After an analysis of the RNA-seq data, we found out that we actually got a contamination by a bacterium called Pseudomonas fluoresense. We assume that this contamination is due to the kluldge. We are not certain of the source of that contamination.
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Latest revision as of 04:00, 18 October 2014

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, the more RNA molecules are present, the more protein will be produced. Using this technology is a chance and we wanted to compare gene expression of Pseudovibrio denitrificans depending on different conditions. These conditions are: M9 media as a reference, or M9 media supplemented with either cadmium, nitrite, or lead. By comparing the gene expressions between conditions and the reference we should be able to find specific highly overexpressed gene.
By 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 could find promoters that can be used to build new biological sensors. These sensors would be created by taking 500 bp upstream these overexpressed genes. The promoters would be characterized adding a reporting gene as the GFP to quantify the expression depending on different concentrations of the compounds used for the RNA-seq. RNA-seq is a critical technique because of the RNA stability. Also, each sample preparation must be done in the exact same conditions than the others to be properly compared.

Pseudovibrio cultures growth in the different conditions were measured by using a Tecan (Infinite M200).
The compounds we tested were nitrite, cadmium and lead.
The kinetic parameters were determined during the characterization of the bacterium characterization of the bacteria.
We tested three compounds in marine broth.

lead


nitrite

cadmium

Title: Kinetic of pseudovibrio in Marine Broth (MB) with either lead, nitrite or cadmium. We diluted the bacteria cultures by 1/5 and 1/10.

Because of the marine broth, which is a cloudy rich medium, we were not able to draw conclusions as it prevented to have a correct measurement of the OD.
Due to that problem with the medium, we preferred to use a M9 medium with casamino acids, because it is a clear media.
In order to be able to observe differences in the bacterial growth, we had to cause a stress to the bacteria by adding the compounds of interest.

lead stress

nitrite stress

cadmium stress


Title: Stress of pseudovibrio denitrificans with differents concentrations of compounds.

The compounds were added after two hours. After analysis of the data, the concentrations we decided to test for the RNA-seq were:
  • 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 protocol of RNA extraction from Tolonen Lab.

Throughout of the experiment, electrophoresis gels are done in order to confirm which have always RNA.


Gel1 Gel2

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.

result1 result2 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. Gel3 Gel4 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.
After an analysis of the RNA-seq data, we found out that we actually got a contamination by a bacterium called Pseudomonas fluoresense. We assume that this contamination is due to the kluldge. We are not certain of the source of that contamination.

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