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population of marine environments due to the higher availability of the chloride and bromide species

Antibacterial Assay

The activity assay was performed against E. coli ATCC 25922 applying microbroth dilution assay as recommended by Clinical Laboratory Standards Institute CLSI (former NCCLS) The tested bacteria were incubated in the MH broth. The assays were performed on 96-well plates, in triplicate for each tested compound, in the concentrations of 250, 125, 62.5, 31.2, 15.6, 7.8 and 3.9 µg/mL. Bioactivity was recorded as blue coloration in the wells accordingly the cells viability after resazurin dye reaction. Tetracycline was used as positive control and pure DMSO (Merck, Darmstadt, Germany) as negative control.

PCR Amplification

Degenerated oligonucleotides were designed to locate the halogenase gene in total DNA of T. wortmannii. They were derived from four conserved motifs present in the amino acid sequences of known eukaryotic halogenases proteins. These oligonucleotides were used to attempt PCR amplification using total DNA from T. wortmannii as the template. The sequences of two sense oligonucleotides were J5 5′-GTGGTTGGTGGTGGCCCTGGAGGG-3′, J6 5′-TCGACCGCTGGCATCGACCAA-3′, J7 5′-TGGGCATGGTTCATTCCTCTCCACAAC-3′ and J8 5′-CCCAGATGAGAAGAATGGGTCAATGAA-3′. PCR mixtures contained 50 ng of template DNA, 50 pmol of each primer, 1 mM MgCl2, 2.5 U Taq DNA Polymerase (MBI Fermentas), 2.5% DMSO and 0.2 mM dNTP-mix. Amplification was obtained by standard procedure with an annealing temperature of 50 °C.