WB800枯草芽孢杆菌 BioVector NTCC质粒载体菌种细胞基因保藏中心

WB800枯草芽孢杆菌

WB800  Bacillus subtilis strain

Genotype:

1. Preparation of competent Bacillussubtilis cells

-overnight culture of the appropriate recipient cells in 5 ml HS medium at 37 °C

-inoculate 50 ml HS medium with 0.5 ml of the overnight culture; incubate undervigorous shaking at 37 °C,record the growth curve

-take samples of 10 ml each when cells reach the stationary phase at 15 minintervals,add 1 ml of sterile glycerol (87%), mix and leave for 15 min on ice

-fractionate into 1 ml aliquots, freeze in liquid nitrogen and store at -80 °C

-check one aliquot from each time point with a reference plasmid DNA (see below)to

identifythe time point(s) yielding high level competent cells; discard the non- orlowcompetent aliquots

2. Transformation of competent Bacillussubtilis cells

-thaw one aliquot at 37 °C,use these cells to inoculate 20 ml LS medium

-shake cells slowly in a 30 °C water bath to obtain maximal competence (about 2h)

-take 1 ml aliquots into a glass tube, add 10 μl of 0.1 M EGTA and incubate for5 min at room temperature

-add plasmid or chromosomal DNA and incubate for 2 h at 37 °C while shaking

-transfer cell suspension into an Eppendorf tube, centrifuge, discardsupernatant

carefullyand resuspend the cells into the final supernatant remaining on the pellet

-plate on selective medium

3. Media and solutions

10x S-base (Spizizen’s salt)

2g (NH4)2SO4

14g K2HPO4

6g KH2PO4

1g sodium citrate

adddistilled water at 100 ml and autoclave,add 0.1 ml 1M MgSO4 after autoclaving

HS medium

66.5ml distilled water, 10.0 ml 10x S-base

2.5ml 20% (w/v) glucose, 5.0 ml 0.1% (w/v) L-tryptophan

1.0ml 2% (w/v) casein, 5.0 ml 10% (w/v) yeast extract (Difco)

10.0ml 8% (w/v) arginine, 0.4% histidine

autoclaveall components separately, tryptophan solution: sterile filtration

LS medium

80.0ml distilled water,10.0 ml 10x S-Base,2.5 ml 20% (w/v) glucose

0.5ml 0.1% (w/v) L-tryptophan,0.5 ml 2% (w/v) casein,5.0 ml 2% (w/v) yeast extract(Difco),0.25 ml 1M MgCl2,0.05 ml 1 M CaCl2

0.1 M EGTA

dissolve3.8 g EGTA in 50 ml distilled water

adjustpH 7.2 using 10 N NaOH,add distilled water to 100 ml

autoclave

BioVector NTCC质粒载体菌种细胞基因保藏中心