Editing Bacillus sub Expression (section)

=== <span style="line-height: 1.5em;">Preparation of competent cells</span>  ===

<span style="line-height: 1.5em;">Sprea </span><span style="line-height: 1.5em;">B. subtilis DB104 cells&nbsp; a LB agar plate&nbsp;without antibiotics (37 °C; for 9 h). </span><br> 

<span style="line-height: 1.5em;">Inoculate </span><span style="line-height: 1.5em;">antibiotic-free SM1&nbsp;media&nbsp; by transferring a single colony and&nbsp;subsequent cultivation (37 °C; 250 rpm for 14–16 h). &nbsp;</span><br> 

<span style="line-height: 1.5em;">Dilute vernight culture&nbsp; in SM1 medium by adjusting&nbsp;optical density at 600 nm (OD 600 ) to 0.5 (approximately&nbsp;2.9×10 7 cells/ml) in a volume of 10 ml and incubated (37 °C; 200 rpm for 3 h).</span> 

<span style="line-height: 1.5em;"> The cell culture volume is doubled by&nbsp;addition of SM2 medium and subsequently supplemented&nbsp;with varied histidine concentrations (final concentration 0,&nbsp;10, 50, 200, 500, and 1,000 μg/ml; total volume 20 ml) and&nbsp;incubate (37 °C; 300 rpm for 2 h). After the treatment, B.&nbsp;subtilis DB104 cells are competent for approximately 1 h.&nbsp;Histidine concentration of 200 μg/ml (final concentration) is used in final protocol.&nbsp;</span> 

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