Part:BBa_K1106008:Experience
路 Inoculated wells with Rhizobium leguminosarum, the positive control, and the E.coli carrying LuxI sequence that was previously induced with 1000 ppb of arsenite, shown positive results.
路 The negative control without LuxI coding sequence and the E.coli carrying LuxI sequence without the addition of arsenite, shown negative results.
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iGEM Buenos_Aires 2013
Method In order to asess if LuxI generator under arsenic sensitive promoter synthesizes AHL (a quorum sensing molecule) when is induced with arsenite, a top agar petri dish was prepared. A layer of sterile LB-agar 1,5% was poured and on top of it an additional layer of LB-agar 0,5% mixed with Chromobacterium v., a bacteria that in presence of certain AHLs turns purple due to the production of violacein. Wells of a volume around 50 ul were performed on the top layer to inoculate different cultures into them. Besides, four different cultures were grown overnight: 路 A Rhizobium leguminosarum culture, grown at 30 潞C in TY medium, as a positive control. 路 Two E. coli cultures (DH5alpha) harbouring the LuxI enzyme gene of Vibrio fischeri, under the control of an arsenite inducible promoter. One of them was added with 1000 ppb of arsenite. 路 An E. coli culture without the codifying sequence of LuxI, as a negative control. Finally, the petri dish, with Chromobacterium v. on the top layer, was inoculated into the wells with samples of 50 ul of each culture and grown overnight at 30潞C. As Chromobacterium v. is a lactone sensitive bacteria, this method allows to detect AHL presence in an easy and very intuitive way by observing the appearance of a violet halo around the inoculation wells which means a positive result.
Results
路 Inoculated wells with Rhizobium leguminosarum, the positive control, and the E.coli carrying LuxI sequence that was previously induced with 1000 ppb of arsenite, shown positive results. 路 The negative control without LuxI coding sequence and the E.coli carrying LuxI sequence without the addition of arsenite, shown negative results.