Transformation of B. subtilis

Transformation Procedure Using B. subtilis RIK1285
IX-1-1. Preparation of Reagents
1) Prepare the following reagents and sterilize by autoclaving.
・ SP I salts (Per 1 L)

(NH4)2SO4, 2 g
K2HPO4, 14 g
KH2PO4, 6 g
Na-Citrate･2H2O, 1 g
MgSO4･7H2O, 0.2 g

・ Casamino Acids/Yeast Extract (Per 100 ml)

Casamino Acids, 2 g
Yeast Extract, 10 g

・ 50％ Glucose
・ 50 mM CaCl2
・ 250 mM MgCl2
・ 100 mM EGTA {ethylene glycol bis (β-amino ethyl ether)-N,N,N',N'-tetraacetic acid}
Adjust to pH 7.0 with NaOH.

2) Using sterile technique, mix the reagents prepared in Step 1 above to the
composition shown below.

・ SP I medium

SP I salts 10 ml
50％ glucose 100 μl
Casamino Acids/Yeast Extract 100 μl

・ SP II medium

SP I medium 5 ml
50 mM CaCl2 50 μl
250 mM MgCl2 50 μl

Method for Preparing B. subtilis RIK1285 Competent Cells
1） Spread a suitable amount of glycerol stock of the host strain on an LB plate and incubate at 37℃ overnight (approximately 16 hours).

2） Inoculate 2 ml of LB medium with a loop full of the host strain incubated overnight on the LB plate. Grow the culture overnight (for approximately 16 hours) at 28℃, shaking at 150 - 180 rpm.

3） Add 50 μl of the culture broth to 5 ml of SP I medium, and incubate at 37℃ at 170 - 180 rpm.

4） Measure OD660 every 30 minutes beginning 1 hour after the start of culturing, and stop culturing once the culture has entered the plateau phase (Fig. 4: near arrow).＊1

＊ 1: Ordinarily, the plateau phase is reached within approximately 5 hours.

5） Add 0.5 ml of the culture broth to 4.5 ml of SP II medium, and incubate at 37℃, 90 - 100 rpm for 90 minutes.

6） Add 50 μl of 100 mM EGTA to the culture medium and shake at 37℃, 90 - 100 rpm for 5 - 10 minutes.

7） Divide the culture among 14 ml round-bottomed test tubes, dispensing 300 μl into each, and transform immediately.＊2

＊ 2: Once prepared, the competent cells cannot be stored.
Use immediately after preparation.

Transformation Method
1） Add 1 μg of DNA solution per 300 μl of the competent cells prepared in IX-1-2.
2） Incubate at 30℃, 90 - 100 rpm for 90 minutes.
3） Spread culture broth on LB plates containing kanamycin (10 μg/ml). Incubate overnight at 37℃.＊1, 2
＊ 1: The transformation efficiency will differ according to the target genes that are inserted. If a sufficient number of colonies cannot
be obtained by plating 300 μl of competent cells, perform transformation by plating with a volume equivalent to (1 μg of DNA solution per 300 μl of competent cells) x n so as to obtain a sufficient number of colonies for screening.

＊ 2: Ordinarily, the transformation efficiency obtained using 1 μg of pBE-S DNA is 3 to 8 x 102 colonies/μg.