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Functional complementation analysis

SS Savvas Saouros
TM Thotegowdanapalya C. Mohan
CC Cristina Cecchetti
SL Silke Lehmann
JB Joseph D. Barrit
NS Nicola J. Scull
PS Paul Simpson
YA Yilmaz Alguel
AC Alexander D. Cameron
AJ Alexandra M. E. Jones
BB Bernadette Byrne
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pDDGFP2 vectors containing the genes encoding WT AtBOR1, WT OsBOR3 and OsBOR3∆1–642 were transformed into a S. cerevisiae ∆bor1p strain for functional complementation analysis as previously described33. As a negative control empty pDDGFP2 vector was also transformed into the S. cerevisiae ∆bor1p strain. Transformants were incubated in 10 mL -URA with 2% (w/v) glucose at 30 °C and 300 rpm shaking overnight. A fivefold serial dilution was carried out with cells starting at OD600 0.5 in -URA media. 10 μL of each dilution was spotted on -URA plates with 2% (w/v) galactose and supplemented with the following concentrations of boric acid: 0, 5, 7.5 mM. Plates were incubated for 30 °C for 5 or 7 days and imaged using a ChemiDoc MP Imaging System (Bio-Rad).

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