Team:Evry/Protocols/09

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<h1> Genomic DNA extraction </h1>
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<h1> Siderophore detection </h1>
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<h2> Aim </h2>
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<h2> Goal </h2>
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<p>.</p>
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<p>Once our bacteria is transformed with the plasmid with the Fur Binding Site and Lac I and the two plasmids with Enterobactins genes, we need to check if our system really work. The production of siderophores can be detected visualy with Blue Agar Chrome Azurol S (CAS). Without siderophore in the medium, CAS and Hexadecyltrimethylammonium bromide  (HDTMA) complexes with ferric iron, producing a blue color. When a bacteria strain produce siderophore, the medium color change from blue to orange.
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</p>
<h2> Preparation </h2>
<h2> Preparation </h2>
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<i>Protocol adapted from Thermo Scientific Genomic extraction notebook</i><br><br>
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<i>Protocol adapted from Louden, B.C., Haarmann, D., and Lynne, A. (2011). Use of Blue Agar CAS Assay for Siderophore Detection.
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<sup>1</sup> </i>
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<p><h3> Blue Dye</h3>
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<b>Solution 1</b><br>
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Dissolve 0,06 g of CAS in 50 mL of distilled water.<br><br></p>
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<b><p>1. Cell culture</b><br>
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<p><b>Solution 2</b><br>
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Cultivate cells in LB medium overnight.<br><br></p>
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Dissolve 0,0027 g of FeCl<sub><small>3</sub></small>-6H<sub><small>2</sub></small>0 in 10 mL of 10 mM HCl.<br>
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<p><b>Solution 3</b><br>
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Dissolve 0,073 g of HDTMA in 40 mL of distilled water.<br></p>
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<p><b>2. Cell harvesting</b><br>
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<p><b>Mix</b><br>
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Set saturated E.coli LM culture into 2 mL tubes. Centrifuge at 8 000 x g for 5 minutes. Discard as much as supernatant as possible.<br><br><p>
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Mix solution 1 with 9 mL of solution 2. Then mix with solution 3.<br>
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Solution should have a blue color. <br>
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Autoclave and store in a bottle. <br></p>
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<p><b>3. Cell lysis</b> <br>
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<h3> Mixture solution</h3>
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Add 180 μL of Digestion Solution and 20 μL of Proteinase K Solution. Resuspend the cells thoroughly with a vortex or a pipette.
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<br>
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Incubate the tubes at 56°C while vortexing occassionally until the cells are completely lysed(~30 minutes).<br>
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Add 20 μL of RNase A solution, mix by vortexing and incubate the tubes for 10 minutes at room temperature.<br>
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<p><b>Minimal Media 9 (MM9) Salt Solution Stock</b><br>
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Add 200 μL of Lysis Solution to the sample. Mix thoroughly by vortexing until a homogeneous mixture is obtained. (~15 secondes)<br>
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Dissolve 15 g of KH<sub><small>2</sub></small>PO<sub><small>4</sub></small>, 25 g of NaCl and 50 g of NH<sub><small>4</sub></small>Cl in 500 mL of distilled water.<br></p>
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Add 400 μL of 50% ethanol and mix with a vortex or a pipette.<br>
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<br>
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<p><b>4. DNA Binding<br></b>
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<p><b>NaOH Stock</b><br>
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Transfer the lysate to a GeneJET Genomic DNA Purification Column inserted in a collection tube. Centrifuge the column at 12 000 x g for 1 minute.<br> Discard the collection tube containing flow-through solution and place the column into a new 2 mL tube.<br>
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Dissolve 25 g of NaOH in 150 mL of distilled water.<br>
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<br>
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pH should be around 12.<br></p>
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<p><b>5. Membrane washing<br></b>
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Add 500 μL of Wash Buffer I (previously added with ethanol). Centrifuge at 1 600 x g. Discard the flow-through and place the purification column back into the collection tube.
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Add 500 μL of Wash Buffer II (previously added with ethanol)to the purification column.
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<p><b>20% Glucose Stock</b><br>
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<br><br>
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Dissolve 20 g of glucose in 100 mL of distilled water.<br></p>
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<p><b>6. Dry membrane<br></b>
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Centrifuge at 16 000 x g for 3 minutes.
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<br><br>
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<p><b>Casamino Acid Solution</b><br>
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<p><b>7. DNA Elution<br></b>
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Dissolve 3 g of Casamino acid in 27 mL of distilled water.<br>
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Add 200 μL of Elution Buffer to the center of the purification column membrane to elute genomic DNA. Incubate for 2 minutes at room temperature and centrifuge at 8 000 x g for 1 minute.
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Extract with 3% 8-hydroxyquinoline in chloroform to remove iron.<br>
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Discard the purification column and store the purified DNA in TrisHCl at -20°C or use it immediatly.
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Filter with a 0,22 µm millipore.</p>
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<h3> CAS agar preparation</h3>
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<p>Add 100 mL of MM9 salt solution to 750 mL of distilled water.<br>
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Bring pH up to 6 and dissolve 32,24 g of piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES); PIPES will not dissolve between pH of 5.<br>
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Add 15 g of Bacto Agar.<br>
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Autoclave and the cool to 50°C.<br>
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Add 30 mL of sterile Casamino acid solution and 10 mL of sterile 20% glucose solution to MM9/PIPES mixture.<br>
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Slowly add 100 mL of Blue Dye solution along the glass wall while mixing thoroughly.<br>
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</p>
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<h3> Bacteria </h3>
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<div id="citation_box">
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<p id="references">References:</p>
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<ol>
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  <li>Louden, B.C., Haarmann, D., and Lynne, A. (2011). Use of Blue Agar CAS Assay for Siderophore Detection. Journal of Microbiology & Biology Education 12,.
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</li>
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</ol>
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</div>
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</p>
</div>
</div>
</div>
</div>

Latest revision as of 09:26, 5 September 2013

Iron coli project

Siderophore detection

Goal

Once our bacteria is transformed with the plasmid with the Fur Binding Site and Lac I and the two plasmids with Enterobactins genes, we need to check if our system really work. The production of siderophores can be detected visualy with Blue Agar Chrome Azurol S (CAS). Without siderophore in the medium, CAS and Hexadecyltrimethylammonium bromide (HDTMA) complexes with ferric iron, producing a blue color. When a bacteria strain produce siderophore, the medium color change from blue to orange.

Preparation

Protocol adapted from Louden, B.C., Haarmann, D., and Lynne, A. (2011). Use of Blue Agar CAS Assay for Siderophore Detection. 1

Blue Dye

Solution 1
Dissolve 0,06 g of CAS in 50 mL of distilled water.

Solution 2
Dissolve 0,0027 g of FeCl3-6H20 in 10 mL of 10 mM HCl.

Solution 3
Dissolve 0,073 g of HDTMA in 40 mL of distilled water.

Mix
Mix solution 1 with 9 mL of solution 2. Then mix with solution 3.
Solution should have a blue color.
Autoclave and store in a bottle.

Mixture solution

Minimal Media 9 (MM9) Salt Solution Stock
Dissolve 15 g of KH2PO4, 25 g of NaCl and 50 g of NH4Cl in 500 mL of distilled water.

NaOH Stock
Dissolve 25 g of NaOH in 150 mL of distilled water.
pH should be around 12.

20% Glucose Stock
Dissolve 20 g of glucose in 100 mL of distilled water.

Casamino Acid Solution
Dissolve 3 g of Casamino acid in 27 mL of distilled water.
Extract with 3% 8-hydroxyquinoline in chloroform to remove iron.
Filter with a 0,22 µm millipore.

CAS agar preparation

Add 100 mL of MM9 salt solution to 750 mL of distilled water.
Bring pH up to 6 and dissolve 32,24 g of piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES); PIPES will not dissolve between pH of 5.
Add 15 g of Bacto Agar.
Autoclave and the cool to 50°C.
Add 30 mL of sterile Casamino acid solution and 10 mL of sterile 20% glucose solution to MM9/PIPES mixture.
Slowly add 100 mL of Blue Dye solution along the glass wall while mixing thoroughly.

Bacteria

References:

  1. Louden, B.C., Haarmann, D., and Lynne, A. (2011). Use of Blue Agar CAS Assay for Siderophore Detection. Journal of Microbiology & Biology Education 12,.

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