Editing Bacillus sub Expression (section)

== Transformation Protocol 2  ==

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

<span style="line-height: 1.5em;">
'''Starvation medium 1 (SM1)''' contains</span> 

*<span style="line-height: 1.5em;">0.2% ammonium&nbsp;sulfate</span> 
*<span style="line-height: 1.5em;">1.4% dipotassium hydrogen phosphate</span> 
*<span style="line-height: 1.5em;">0.6% potassium dihydrogen phosphate</span> 
*<span style="line-height: 1.5em;">0.07% sodium citrate</span> 
*<span style="line-height: 1.5em;">0.5%&nbsp;glucose</span> 
*<span style="line-height: 1.5em;">0.02% magnesium sulfate heptahydrate</span> 
*<span style="line-height: 1.5em;">0.2% yeast </span>extract

*<span style="line-height: 1.5em;">0.025% casamino acids. </span><br>

Mix all components of SM1 medium and autoclave together. <br> 

'''<span style="line-height: 1.5em;">Starvation medium 2</span>''' (SM2)'''<span style="line-height: 1.5em;"> is less rich in nutrients and includes </span>''' 

*<span style="line-height: 1.5em;">0.2% ammonium&nbsp;sulfate, </span> 
*<span style="line-height: 1.5em;">1.4% dipotassium hydrogen phosphate, </span> 
*<span style="line-height: 1.5em;">0.6% potassium dihydrogen phosphate, </span> 
*<span style="line-height: 1.5em;">0.07% sodium citrate, </span> 
*<span style="line-height: 1.5em;">0.5%&nbsp;glucose, </span> 
*<span style="line-height: 1.5em;">0.08% magnesium sulfate heptahydrate, </span> 
*<span style="line-height: 1.5em;">0.1% yeast
</span>

extract, 

*<span style="line-height: 1.5em;">0.01% casamino acids</span> 
*<span style="line-height: 1.5em;">0.05% calcium chloride </span><br>

<span style="line-height: 1.5em;">Mix all components of SM2 medium&nbsp; and autoclave together. </span><br> 

<span style="line-height: 1.5em;">Histidine solution was sterilized by filtration using 0.2-μm filters</span> 

<br> 

=== <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> 

<br> 

=== Transformation procedure  ===

<span style="line-height: 1.5em;">Five hundred microliters of the competent cells is mixed&nbsp;with varied amounts of plasmid DNA (pHY300Car—2, 5,
10, 20, 40, 100, 250, 500, 1,000 ng) and incubate (37 °C;&nbsp;200 rpm for 30 min). In order to recover the cells, 300 μl of
fresh LB medium is supplemented and competent B. subtilis DB104 are additionally incubated (37 °C; 200 rpm for
30 min). Two hundred microliters of B. subtilis DB104 cell&nbsp;suspension is subsequently spread on LB agar plates with
selective antibiotic (tetracycline, final concentration&nbsp;15 μg/ml).</span> 

=== Transformation Protocol 3  ===

<span style="line-height: 1.5em;">An overnight culture was grown in minimal medium with 2.5 μg/ml erythromycin at 37°C, with continuous shaking at 300 rpm. After 100-fold dilution of the overnight culture in minimal medium, incubation was continued, and the optical density at 600 nm (OD600) was monitored. Two hours after the transition from exponential to stationary-phase growth, 1 μg of DNA was added to 0.5 ml of competent cells. Samples were kept at 37°C with shaking. After 20 min, 0.3 ml of TY medium was added, and growth was continued for another 30 min, after which the cells were plated on selective TY-agar plates.</span><br> 

=== Transformation Protocol 4  ===

==== Media Preparation  ====

''10X Medium A base:'' 

*Yeast extract 10g 
*Casamino acids 2g 
*Distilled water to 900mL 
*Autoclave, then add&nbsp;: 
*50% glucose, filter sterilized 100mL

''10X Bacillus salts:'' 

*(NH4)2SO4 20g 
*Anhydrous K2HPO4 139.7g 
*KH2PO4 60g 
*Tri-sodium citrate 10g 
*MgSO4•7H2O 2g 
*SDW to 1000mL

''Medium A'' 

*Sterile water 81mL 
*10X Medium A base 10mL 
*10X Bacillus salts 9mL 
*L-Tryptophan (11mg/mL) 0.1mL

''Medium B'' 

*Medium A 10mL 
*50mM CaCl2•2H2O 0.1mL 
*250nM MgCl2•6H2O 0.1mL

''Important:'' 

*Autoclave Medium A base before adding glucose, and autoclave Bacillus salts 
*Store aliquots of 10X Medium A base 10mL and 10X Bacillus salts 9mL and keep them in the fridge, never use them twice to avoid contamination

<br> 

==== Making Bacillus competent  ====

#Grow one blank plate of ''Bacillus subtilis'' (or several if you want to transform different strains) for 20 hours at 37ºC (plate been kept on the bench for several days would be better) 
#Inoculate about 12mL of medium with several colonies. Mix the contents of the tube. Check with OD650. Start OD should be between 0.1 and 0.2. Be careful to pipette 0.8mL of this mixture into the cuvette to measure and dispose of it after measurement to avoid contamination in the main mixture. 
#Incubate at 37ºC with vigorous shaking. Read the OD650 every 20min (never keep the solution you used for measuring!) 
#Plot log(OD650) in function of time. After a brief lag, you should observe a exponential increase. After awhile, it will leave the exponential growth; the moment at which it leaves the exponential path is denoted as t0 (3 on the graph). It should take about 100min and the OD should be between 0.35 and 0.55.

OD650 of B.subtilis 

#At t0, incubate for 90 minutes at 37ºC with vigorous shaking. 
#Transfer 0.05mL of this culture into 0.45mL of pre-warmed Medium B in an Eppendorf tube. You have to prepare one tube for each transformation, plus an extra tube for a DNA-less control. 
#Incubate the diluted cultures at 37ºC with shaking for 90min. At this moment, the cells are HIGHLY COMPETENT. 
#To check for competency, you can look at cells under the microscope; competent cells are very motile.

=== Transforming  ===

#Spin Eppendorf tubes containing cells. Remove 400µL of liquid to keep only 100µL of the culture (to concentrate cells). Re-suspend the cell pellet in the remaining culture. 
#To transform from competent glycerol stocks, spin the tube at about 1600rpm for 20min, remove the supernatant (glycerol), and add 100µL of pre-warmed medium B. 
#Mix the cells thoroughly. 
#Add 0.6µg of DNA to the competent cells. 
#Incubate for 30min at 37ºC with shaking. 
#Plate 100µL of transformed cells onto selective agar.

=== Glycerol stocks  ===

#To freeze competent Bacillus cells, spin down the fresh competent cells to obtain a pellet. 
#Remove all supernatant. 
#Re-suspend cells in 500µL 60% glycerol. 
#Freeze tubes at -80ºC.