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Applications of BBa_K2139004

C. crescentus was inoculated - in triplicates, in PYE media supplemented with varying concentrations (0 to 10mM) of CuSO4. The cultures were incubated at 30C for 72 hours, while shaking. The OD600 was measured to determine concentrations of CuSO4 that would inhibit Caulobacter growth.

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Figure 1. Copper toxicity test. A) Growth measurements of p4A723 Caulobacter cultures grown for 3 days - in triplicates, in PYE supplemented with 0, 0.0001, 0.001, 0.01, 0.5, 1 and 10mM CuSO4. B) Growth measurements of sLac Caulobacter in PYE supplemented with 0.1, 0.2, 0.3, 0.4, 0.5 mM CuSo4 to more precisely determine toxic concentration of copper.

C. crescentus encoding sLAC in p4A723 and just p4A723 (as a negative control) were inoculated into flasks containing 15 mL cultures of PYE in the presence or absence of 300 µM CuSO4. The cultures were grown for 3 days, OD600 was normalized and ABTS assay was performed in 96-well plates. The protocols were derived from More et al(2011). Specifically, 176 µl of Sodium Acetate (pH 5), 10 µl of a culture, 4 µl of 0.5 M CuSO4 (10 mM CuSO4 final concentration) and 10 µl of 10 mM ABTS (0.5 mM ABTS final concentration) were added to each well. The plate was then incubated at 30C for an hour and absorbance at 420nm was measured using a Varioskan plate reader.

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Figure 2.Results of ABTS assay on sLac and p4A723 cultures. The test was performed with and without addition of copper.

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