Dandekar & Chandler:Transformation into amyE

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Transformation into AmyE

Transformation of Bacillus subtilis

Modified from A.L. Sonenshein, contributed by Thao T.

Materials

SpC medium

10 mL 1x T base:

• 2 g (NH)2SO4
• 14 g K2HPO4
• 6 g KH2PO4
• 1 g Na3-citrate-6H2O
• 1 liter H2O

• 0.1 mL 50% glucose
• 0.1 mL 2% MgSO4
• 0.25 mL 1% casamino acids
• 0.2 mL 10% nutrient broth
• 0.1 mL 0.5% tryptophan
• 0.05 mL 1% phenylalanine

SpT medium

• 10 mL 1x T base
• 0.1 mL 50% glucose
• 0.41 mL 2% MgSO4
• 0.1 mL 1% casamino acids
• 0.1 mL 10% nutrient broth
• 0.1 mL 0.5% tryptophan
• 0.05 mL 1% phenylalanine

Recipient strain

Transforming DNA

Methods

1. The evening before the transformation, streak the recipient strain on rich medium (LB) and grow up at 30C overnight.
2. Using the overnight plate culture, inoculate 10 mL SpC medium in a 125 mL baffled flask to a density of 5 x 10⁷ cells/mL (OD600 = 0.2-0.4). Shake the culture at 37C, 275-300 rpm until the culture reaches early stationary phase.
   1. For B. subtilis JH642, we monitored growth every 30 minutes until it began to level off (approximately 3.5 hours to OD 1.15)
3. Dilute the SpC culture by adding 2 mL to 10 mL fresh SpT medium. Shake at 37C for 70-90 minutes at 250-275 rpm.
4. Continue immediately with transformation or freeze competent cells.
   1. To freeze cells: combine 500 μL cells and 100 μL 50% glyerol in an eppendorf tube. Store at -80C.
5. Transfer 1 mL aliquots of competent cells to eppendorf tubes.
6. Designate one tube a negative (no DNA) control. Add transforming DNA to other tubes. In general 1-3 μL of a DNA prep is sufficient.
   1. We used 2 μL of ~70 ng/μL pDR111.
7. Shake tubes at 37C for 1 hour.
   1. Bacillus guide recommends using large test tubes because Bacillus likes to be aerated.
8. Plate 50-250 μL on selective medium and incubate overnight at 30C or 37C.

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Getting stuff on the chromosome

We used pDR111, a plasmid with a specR marker that integrates into the amyE locus. This is a nonessential gene that confers the ability to degrade starch. Make competent B. subtilis (as above), transform, select on LB + Spec100. To screen for successful clones:

1. Grow stabs of candidates overnight at 37C on a starch plate:
   1. 33 grams tryptose blood agar base (TBAB)
   2. 10 grams starch (1% wt/vol; ARGO brand pure corn starch)
   3. QS to 1 liter
   4. Dissolve media on heated stir plate, autoclave 30 min to sterilize
   5. Solidified plates will look cloudy
2. Flood the plate with iodine or wescodyne and observe immediately (false positives may arise with time). If there is clearing around the colony, it can degrade starch and crossover was unsuccessful. It may be easier to see clearing zones from the bottom of the plate. We used WT B. subtilis as a positive control and E. coli as a negative control.
3. Verify using primers to downstream of AmyE (genomic) and from within the plasmid (specR).
   1. Colony PCR is difficult, so we prepared genomic DNA using a general Gram - protocol (Gentrakit 158388)