TITLE: Transformation of heterolous DNA in Bacillus subtilis
AUTHORS: Kristoffer Bach Falkenberg, Cristina Hernandez Rollan, Maja Rennig, Andreas Birk Bertelsen, Morten Norholm

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">B. subtilis </span><span>is a gram-positive bacteria used by both academia and industry as a protein production workhorse. This is due to its' excellent fermentation properties, high production titers, and capacity to secrete proteins into the extracellular medium.</span></div><div class = "text-block"><span>This protocol describes transformation of </span><span style = "font-style:italic;">B. subtilis </span><span>by natural competence. The method utilizes the natural stress-induced competence of </span><span style = "font-style:italic;">B. subtilis</span><span> to take up heterologous DNA. The protocol requires the cells to be grown for a specific amount of time in starvation media (SM). The protocol is adapted from Vojcic, L., Despotovic, D., Martinez, R., Maurer, K., & Schwaneberg, U. (2012). An efficient transformation method for </span><span style = "font-style:italic;">Bacillus subtilis</span><span> DB104. </span><span style = "font-style:italic;">Applied Microbiology and Biotechnology</span><span>, </span><span style = "font-style:italic;">94</span><span>(2), 487–493. https://doi.org/10.1007/s00253-012-3987-2.</span></div></div>

[STEPS]
?. [Preparation of stock solutions]
Mix 10xSM1 stock
?. [Preparation of stock solutions]
Mix 1xSM2 stock
SM2 is made as a 1x stock since it otherwise precipitates
?. [Preparation of stock solutions]
Weigh the following in a 100mL blue cap bottle:2 g ammonium sulphate14 g dipotassium hydrogen phosphate6 g potassium dihydrogen phosphate0.7 g sodium citrate0.2 g magnesium sulfate heptahydrate2 g yeast extract0.25 g casamino acids
?. [Preparation of stock solutions]
Add MQ water to  and mix to dissolve the ingredients
100 mL
?. [Preparation of stock solutions]
Weigh the following in a 1L blue cap bottle:2 g ammonium sulfate14 g dipotassium hydrogen phosphate6 g potassium dihydrogen phosphate0.7 g sodium citrate0.8 g magnesium sulfate heptahydrate1 g yeast extract0.1 g casamino acids
?. [Preparation of stock solutions]
Add MQ water to  and mix to dissolve the ingredients
1 L
?. [Preparation of stock solutions]
Autoclave the stocks and store at  or
0 Room temperature
4 °C
?. [Overnight culture]
Prepare fresh SM1 media from the stock by mixing 1 part 10xSM1 media stock with 9 parts sterile MQ water and 25uL 200g/L glucose stock per mL media (to final concentration of 5g/L).
?. [Preparation of stock solutions]
Prepare a 200g/L glucose and a 1M CaCl2 stock, separately
Glucose is prepared separately to avoid Millard's reaction. CaCl2 is prepared separately since it makes the SM2 stock precipitate.
?. [Transformation]
Measure OD600 of the O/N culture
?. [Overnight culture]
Inoculate 10mL SM1 media in a 50mL falcon tube with a single colony from a freshly streaked plate
?. [Overnight culture]
Incubate  at  with 250RPM shaking
37 °C
Make sure to not incubate the overnight culture for longer than  . Using an overnight culture that has been incubating for longer than this, often results in low transformation efficiencies
?. [Transformation]
Dilute the O/N culture in  SM1 media to a final OD600 of 0.5 in a  Erlenmeyer flask
10 mL
250 mL
?. [Transformation]
Grow culture at  at 250 RPM shaking for
37 °C
?. [Transformation]
Add  SM2 medium to the culture
10 mL
?. [Transformation]
Add  200g/L glucose stock to the culture
250 µl
?. [Transformation]
Swirl the culture around, and add  1M CaCl2 stock while the media is still in motion
45 µl
The swirling is done since high local concentrations of CaCl2 makes the media precipitate. This way the CaCl2 is mixed in the media before it precipitates
?. [Transformation]
Grow culture at  at 250 RPM shaking for
37 °C
After this step the cells are competent for approximately
?. [Transformation]
Distribute the cells in  aliquotes in  eppendorf tubes
500 µl
2 mL
The protocol can be paused at this point, by adding     glycerol to the aliquotes and freezing them in a   freezer. The protocol can be restarted by thawing the cells on ice and moving on the next step. This can also be used to prepared stocks of strains that are used often
?. [Transformation]
Add 250ng - 1pg of plasmid DNA to the aliquotes
Some protocols call for linearized plasmids or PCR products, although we have found that circular  plasmids work as well
?. [Transformation]
Incubate the aliquotes in a thermoblock for  at  with 800 RPM shaking
37 °C
?. [Transformation]
Add  LB media and recover the cells for at least  at  with 800 RPM shaking
300 µl
37 °C
The longer duration of incubation the better
?. [Transformation]
Plate up to  of each aliquot on LB agar plates with appropriate antibiotics
200 µl
In order to increase the number of colonies on the transformation plates, the aliquotes can be centrifuged for   and reinoculated in a smaller volume.
?. [Transformation]
Incubate the plates  at
37 °C