Oct 27, 2020
Transformation of heterolous DNA in Bacillus subtilis
- Kristoffer Bach Falkenberg1,
- Cristina Hernandez Rollan1,
- Maja Rennig1,
- Andreas Birk Bertelsen1,
- Morten Norholm1
- 1Technical University of Denmark
Protocol Citation: Kristoffer Bach Falkenberg, Cristina Hernandez Rollan, Maja Rennig, Andreas Birk Bertelsen, Morten Norholm 2020. Transformation of heterolous DNA in Bacillus subtilis. protocols.io https://dx.doi.org/10.17504/protocols.io.bdmti46n
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol routinely and it works for us
Created: March 13, 2020
Last Modified: October 28, 2020
Protocol Integer ID: 34195
Keywords: B. subtilis, Bacillus, Bacillus subtilis, Transformation, Natural competence
Abstract
B. subtilis 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.
This protocol describes transformation of B. subtilis by natural competence. The method utilizes the natural stress-induced competence of B. subtilis 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 Bacillus subtilis DB104. Applied Microbiology and Biotechnology, 94(2), 487–493. https://doi.org/10.1007/s00253-012-3987-2.
Guidelines
This protocol works for a range of B. subtilis strains (e.g. 168, WB800, PY79, KO7, KO7-S, and derivatives), although the protocol might need optimization for heavily growth impaired strains. For protein production, it is generally recommended to use a protease deficient strains such as WB700, WB800, KO7 or KO7-S.
Materials
MATERIALS
GlucoseP212121Catalog #Glucose
Sodium citrateP212121
Ammonium SulfateP212121
Potassium phosphate (dibasic)P212121
Calcium Chloride
Potassium dihydrogen phosphateMerck MilliporeSigma (Sigma-Aldrich)Catalog #NIST200B
Magnesium SulfateAmrescoCatalog #0338
Yeast extract
Bacto™ Casamino AcidsThermo FisherCatalog #223020
Safety warnings
This protocol describes the construction of GMO classified organisms. Make sure that the local GMO and safety legislations are respected.
Before start
Make sure you have your recipient strain freshly streaked on an agar plate.
Preparation of stock solutions
Preparation of stock solutions
Mix 10xSM1 stock
Weigh the following in a 100mL blue cap bottle:
- 2 g ammonium sulphate
- 14 g dipotassium hydrogen phosphate
- 6 g potassium dihydrogen phosphate
- 0.7 g sodium citrate
- 0.2 g magnesium sulfate heptahydrate
- 2 g yeast extract
- 0.25 g casamino acids
Add MQ water to 100 mL and mix to dissolve the ingredients
Mix 1xSM2 stock
Note
SM2 is made as a 1x stock since it otherwise precipitates
Weigh the following in a 1L blue cap bottle:
- 2 g ammonium sulfate
- 14 g dipotassium hydrogen phosphate
- 6 g potassium dihydrogen phosphate
- 0.7 g sodium citrate
- 0.8 g magnesium sulfate heptahydrate
- 1 g yeast extract
- 0.1 g casamino acids
Add MQ water to 1 L and mix to dissolve the ingredients
Autoclave the stocks and store at Room temperature or 4 °C
Prepare a 200g/L glucose and a 1M CaCl2 stock, separately
Note
Glucose is prepared separately to avoid Millard's reaction. CaCl2 is prepared separately since it makes the SM2 stock precipitate.
Overnight culture
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).
Inoculate 10mL SM1 media in a 50mL falcon tube with a single colony from a freshly streaked plate
Incubate Overnight at 37 °C with 250RPM shaking
Note
Make sure to not incubate the overnight culture for longer than 16:00:00 . Using an overnight culture that has been incubating for longer than this, often results in low transformation efficiencies
Transformation
Transformation
Measure OD600 of the O/N culture
Dilute the O/N culture in 10 mL SM1 media to a final OD600 of 0.5 in a 250 mL Erlenmeyer flask
Grow culture at 37 °C at 250 RPM shaking for 03:00:00
Add 10 mL SM2 medium to the culture
Add 250 µL 200g/L glucose stock to the culture
Swirl the culture around, and add 45 µL 1M CaCl2 stock while the media is still in motion
Note
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
Grow culture at 37 °C at 250 RPM shaking for 02:00:00
Note
After this step the cells are competent for approximately 01:00:00
Distribute the cells in 500 µL aliquotes in 2 mL eppendorf tubes
Note
The protocol can be paused at this point, by adding 250 µL 50 % volume glycerol to the aliquotes and freezing them in a -80 °C 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
Add 250ng - 1pg of plasmid DNA to the aliquotes
Note
Some protocols call for linearized plasmids or PCR products, although we have found that circular plasmids work as well
Incubate the aliquotes in a thermoblock for 00:30:00 at 37 °C with 800 RPM shaking
Add 300 µL LB media and recover the cells for at least 02:00:00 at 37 °C with 800 RPM shaking
Note
The longer duration of incubation the better
Plate up to 200 µL of each aliquot on LB agar plates with appropriate antibiotics
Note
In order to increase the number of colonies on the transformation plates, the aliquotes can be centrifuged for 6000 x g, 00:02:00 and reinoculated in a smaller volume.
Incubate the plates Overnight at 37 °C