Transformation of Bacillus subtilis

Microbial Genetics@ FCT/UNL

Transformation of Bacillus subtilis 

Select a colony and grow it overnight (37ºC, 150rpm) in 3mL SP medium with the appropriate antibiotics (if applicable);

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Dilute the overnight culture 1:50 in SP medium (200L of culture in 10mL SP) with the appropriate antibiotics;

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Follow the growth at 37ºC and 150rpm until T0 + 60 minutes (usually when O.D.600nm reaches 1.2);

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Add plasmid DNA to 500L of the competent cell culture (the amount of DNA should be 50ng in the 500L);

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Incubate at 37ºC and 150 rpm for 30 minutes (up to 2 hours) and plate 100 to 200L;

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Select transformant cells in LB plates with the appropriate antibiotics.

SP medium for transformation       

18.4mL Phosphate Citrate Buffer – Spizizen’s salts without (NH4)2SO4 1mL Glucose 40% (2% final) 400L Casein Hydrolysate 1% (0.02% final) 100L L-Tryptophan 10mg.mL-1 Spizizens salts without (NH4)2SO4 (1000mL) 10L CAF (Ferric Ammonium Citrate) 22mg.mL-1 Potassium phosphate dibasic (K2HPO4) 14g Potassium phosphate monobasic (KH PO ) 6g 2 4 100L Potassium Glutamate 40% Sodium citrate tribasic 1g 60L MgSO4 1M