"
Team:Evry/Protocols/09
From 2013.igem.org
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. 1Blue Dye
Solution 1Dissolve 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:
- Louden, B.C., Haarmann, D., and Lynne, A. (2011). Use of Blue Agar CAS Assay for Siderophore Detection. Journal of Microbiology & Biology Education 12,.
