Editing Bacillus sub Expression (section)

== B. subtilis Competent Cell Preparation and Transformation  ==

=== Very Important:<br>  ===

<font color="red">ampicillin selection is for E. coli only. The amp gene is not expressed in Bacillus or other Gram-positive organisms. Use erythoromycin (1 μg/ml) with pLIKE-rep or chloramphenicol (5 μg/ml) with pLIKE-int for selection in B. subtilis <br></font> 

<font color="red">'''For pHCMC04: Use&nbsp;5 µg/mL Chloramphenicol for E coli and Bacillus, use ampicillin in E coli'''</font><br><br> 

<br>Yasbin, R. E., G. A. Wilson, and F. E. Young. 1975. Transformation and transfection in&nbsp;lysogenic strains of Bacillus subtilis: Evidence for selective induction of prophage in competent cells. J. Bacteriol. 121:296-304.&nbsp;Zahler, S. A. (personal communication) 

<br>Competence development in B. subtilis is one of several stationary phase processes&nbsp;triggered by a nutritional downshift. Since the pioneering work of Anagnostopolous and Spizizen, a number of protocols for preparing competent B. subtilis cells have appeared. The&nbsp;following method, modified by Ron Yasbin from protocols developed in the Frank Young lab&nbsp;at Rochester, was used routinely in Stan Zahler’s wonderful bacterial genetics course at&nbsp;Cornell. This protocol assumes that you use a spectrophotometer that accepts 16×125 mm&nbsp;test tubes. If your spectrophotometer, like mine, works only with cuvettes, simply increase&nbsp;the culture volume to 10 or 20 ml in a 250-ml Erlenmeyer flask. 

<br>1. Streak recipient strain on one-half of a Tryptose Blood Agar Base plate. Incubate&nbsp;overnight (18 hr) at 37°C. 

<br>2. Inoculate a few colonies into 4.5 ml of Medium A in a 16×125 mm test tube that lacks&nbsp;visible scratches. Mix the contents of the tube thoroughly. Read its optical density at 650 nm in the spectrophotometer. Adjust the OD 650 to be 0.1-0.2, maintaining the volume at&nbsp;4.5 ml. 

<br>3. Incubate at 37°C with vigorous aeration. Read the OD 650 every 20 min, plotting OD 650&nbsp;against time on semi-log paper. After a brief lag, the OD should increase logarithmically—that is, they should fall on a straight line. Note the point at the culture&nbsp;leaves log growth—the graph points fall below the straight line. In B. subtilis genetics, this point is known as t 0 . It should take 60-90 minutes of incubation and occur at&nbsp;OD 650 =0.4-0.6. 

<br>4. Continue incubation for 90 minutes after the cessation of log growth (t 90 ). Transfer 0.05&nbsp;ml of this culture into 0.45 ml of pre-warmed Medium B in a 16×125 mm test tube. Set up one tube for each transformation you intend to perform, plus an extra for a DNA-less&nbsp;control. 

<br>5. Incubate the diluted cultures at 37°C with vigorous aeration for 90 min. At this point,&nbsp;the cultures should be highly competent. 

<br>6. Add 1 μg of DNA to the competent cells and incubate at 37°C with aeration for 30&nbsp;minutes. 

<br>7. Plate aliquots of the transformed cells onto selective agar. 

'''Medium A'''<br>Sterile water: 81 ml<br>10× Medium A base: 10 ml<br>10× Bacillus salts: &nbsp;<span style="line-height: 1.5em;">9 ml</span> 

<span style="line-height: 1.5em;">autoclave</span> 

<br>'''Medium B'''<br>Medium A: 10 ml<br>50 mM CaCl2 ·2H2O: &nbsp;0.1 ml<br>250 mM MgCl2 ·6H2O: &nbsp;<span style="line-height: 1.5em;">0.1 ml</span> 

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'''10× Medium A base''':<br>Yeast extract: &nbsp;10 g<br>Casamino acids: &nbsp;2 g<br>Distilled water to 900 ml<br>50% glucose:&nbsp;100 ml 

<span style="line-height: 1.5em;">Autoclave.</span> 

<br> 

'''10× Bacillus&nbsp;<span style="line-height: 1.5em;">salts</span>''' 

(NH4)2SO4: &nbsp;20 g<br>K2HPO4·3H2O: &nbsp;183 g<br>KH2PO4: &nbsp;60 g<br>Na + citrate: &nbsp;10 g<br>MgSO4·7H2O: &nbsp;2 g<br>Water &nbsp;<span style="line-height: 1.5em;">to 1000 ml</span>