TITLE: Transformation of Bacillus subtilis with DNA obtained from natural donor cell lysis
AUTHORS: James P. Finn IV, Sina A. Riazi, Mitchell T. Armstrong, Briana M. Burton

[DESCRIPTION]
    Natural transformation is a mechanism many bacteria use to acquire free DNA from the environment. Bacillus subtilis (B. subtilis) can perform natural transformation by entering the physiological state of competence, wherein B. subtilis produces the necessary proteins to acquire DNA and subsequently integrate homologous DNA into its genome. This trait is advantageous to researchers, for competence can be induced and utilized for efficient construction of new bacterial strains. For this purpose, the primary source of donor DNA is typically purified DNA in the form of genomic DNA (gDNA) or plasmid DNA. The benefit of using purified DNA is that a new strain can be constructed efficiently without concern for contamination with the donor cells. This involves growing both a donor and recipient strain, inducing competence in the recipient, extracting donor gDNA, and subsequently mixing the gDNA and recipient cells for the transformation to occur. The downside to using purified DNA is the purification process itself. Bacterial gDNA extraction usually involves phenol and chloroform or a commercial nucleic acid extraction kit. The time and resources involved in preparation of purified donor DNA severely limits the number of donors that are possible to prepare and use in a single experiment.
    We propose to exploit natural cell lysis as a new method for transforming B. subtilis to bypass the time-intensive gDNA purifications. Described herein, the donor strain is grown to the point of sufficient, natural cell lysis with minimal post-incubation benchwork to obtain a lysate containing the desired donor DNA. This lysate is used as the donor substrate for a subsequent transformation. The growth and handling of the recipient is the same regardless of the donor DNA source. There are a few major advantages of this method. With this protocol, a researcher can obtain sufficient DNA for a transformation with less than 10 minutes of benchwork, affording the researcher more flexibility in scheduling their day. The utility of the application is further realized with its scalability. The natural donor cell lysis can occur in 96-well plates, and the resource cost of this is minimal compared to performing an equivalent number of gDNA extractions. Moreover, previously frozen lysates can be used as the donor DNA to obtain successful transformants with minimal loss of efficiency. By using cell lysate as the donor DNA source instead of purified DNA, we developed a method that reduces the time at the bench and improves scalability.

[BEFORE_START]
Check the donor and recipient resistance markers to make sure they are compatible.
If selection against donor cells is not possible, then it is necessary to filter the donor DNA crude cell lysate.
It is not necessary to filter the crude lysate from the donor if selection can be used to remove donor bacteria.
If filtration is necessary and many donor samples are to be used, such as in a 96-well plate format, then this will increase the cost of the protocol. Microfuge-based filtration of individual samples was validated here. 96-well plate filtration was not conducted here but also should be compatible with this transformation procedure.

[GUIDELINES]
Sterile technique is important during the protocol.

[STEPS]
SECTION: Two days before the transformation: Preparing donor and recipient strains.
1. Streak out the donor strain onto a LB agar plate with appropriate antibiotics and incubate at 37 °C for approximately 18 hours to obtain fresh, single colonies.
SECTION: Two days before the transformation: Preparing donor and recipient strains.
2. Streak out the recipient strain onto a LB agar plate and incubate at 30 °C for, at most, 42 hours to obtain fresh, single colonies in two days.
SECTION: One day before the transformation: Preparing the donor culture.
3. If the recipient was not streaked out yesterday, then streak out the recipient onto a LB agar plate with appropriate antibiotics and incubate at 37 °C for fresh, single colonies about 18 hours later.
SECTION: One day before the transformation: Preparing the donor culture.
4. In 18mm sterile, glass test tubes, prepare 1 mL for each donor.

SECTION: One day before the transformation: Preparing the donor culture.
5. Inoculate 1 mL with one donor colony.
SECTION: One day before the transformation: Preparing the donor culture.
4.1. Alternatively, standard 96-well plates may be used with 200 µL of 1x MC medium per well or square deep-well, 96-well plates may be used with 400 µL of 1x MC medium per well.

Note: ensure there is enough donor culture for the number of unique transformations.
SECTION: One day before the transformation: Preparing the donor culture.
4.2. Table for making 1 mL or 400 µL of 1x MC medium.
 

SECTION: One day before the transformation: Preparing the donor culture.
6. Vortex at setting 3 to 4 for 10 seconds and remove the inoculating stick.

10 s 
SECTION: One day before the transformation: Preparing the donor culture.
6.1. Alternatively, if using 96-well plates, then use a pipette tip to inoculate each well with a colony.
SECTION: One day before the transformation: Preparing the donor culture.
7. Incubate donor culture at 37 °C with aeration.
Note: 18 mm test tubes can aerate rolling in a roller drum. 
Note: 96-well plates should have an aeration seal applied and aerate on a platform shaker at 350 rpm.
SECTION: One day before the transformation: Preparing the donor culture.
8. Incubate for 20-24 hours to obtain sufficient cell lysis.

1440 min 

SECTION: Day of the transformation: Mixing donor and recipient.
9. Four hours before the end of the 20-24 hours incubation time for the donor, prepare 1 mL for the recipient in sterile, 18mm glass test tubes.
SECTION: Day of the transformation: Mixing donor and recipient.
11. Inoculate 1 mL with one recipient colony using a sterile inoculation stick.
SECTION: Day of the transformation: Mixing donor and recipient.
12. Incubate the recipient cultures at 37 °C for 4 hours with aeration.
Use the same aeration methods as used for the donor cultures.

  

240 min
SECTION: Day of the transformation: Mixing donor and recipient.
13. Shortly before 4 hours have elapsed, prepare the donor for transformation:

Is it necessary to filter the donor? (see Before Start)
SECTION: Day of the transformation: Mixing donor and recipient.
19. Donor DNA source may sit at room temperature while preparing the transformation tubes, but it can be stored at -30 °C.
SECTION: Day of the transformation: Mixing donor and recipient.
10. The recipient can be grown in either glass test tubes or 96-well plates:

Will the recipient be grown in Test Tubes or 96-well plates?
(Select from the first step case below and continue within that step case for the remainder of the protocol.)
SECTION: Day of the transformation: Mixing donor and recipient.
12. Incubate the recipient cultures at 37 °C for 4 hours with aeration.
Use the same aeration methods as used for the donor cultures.

  

240 min
SECTION: Day of the transformation: Mixing donor and recipient.
None. If filtration is necessary (see Before Start):
SECTION: Day of the transformation: Mixing donor and recipient.
None. If working in 18 mm glass test tubes:
SECTION: Day of the transformation: Mixing donor and recipient.
None. Aliquot 1 mL sample of the donor to a 1.7-microfuge tube.
SECTION: Day of the transformation: Mixing donor and recipient.
None. Spin the sample at 21,000 x g in a centrifuge for 1 minute. Remove supernatant to a new microfuge tube. Take care not to disrupt cells and cell debris in pellet.
SECTION: Day of the transformation: Mixing donor and recipient.
None. Filter the supernatant through a 0.22 µm PES filter.

This filtrate is the source of donor DNA.
Note: cellulose acetate and nylon filters are equally effective, but PES is usually most cost effective.
SECTION: Day of the transformation: Mixing donor and recipient.
None. Donor DNA source may sit at room temperature while preparing the transformation tubes.
SECTION: Day of the transformation: Mixing donor and recipient.
None. 
SECTION: Day of the transformation: Mixing donor and recipient.
14. Is the donor growing in test tube or 96-well plate format?
SECTION: Day of the transformation: Mixing donor and recipient.
15. Filter away from the donor bacteria with a 0.22 µm filter type.

This filtrate is the source of donor DNA.
SECTION: Day of the transformation: Mixing donor and recipient.
16. Donor DNA source may sit at room temperature while preparing the transformation tubes, but it can be stored at -30 °C.
SECTION: Day of the transformation: Mixing donor and recipient.
15. Aliquot 1 mL sample of the donor to a 1.7-microfuge tube.
SECTION: Day of the transformation: Mixing donor and recipient.
16. Spin the sample at 21,000 x g (max speed) in a centrifuge for 1 minute.

21000 x g, 1 min 
SECTION: Day of the transformation: Mixing donor and recipient.
17. Remove supernatant to a new microfuge tube. Take care not to disrupt cells and cell debris in pellet.
SECTION: Day of the transformation: Mixing donor and recipient.
18. Filter the supernatant through a 0.22 µm PES filter.

This filtrate is the source of donor DNA.
Note: cellulose acetate and nylon filters are equally effective, but PES is usually most cost effective.
SECTION: Day of the transformation: Mixing donor and recipient.
11. Inoculate each well of a 96-well plate with 200 µL with recipient.

Note: A deep-well, 96-well plate can be used for the recipient growth with 400 µL of 1x MC medium.
SECTION: Day of the transformation: Mixing donor and recipient.
11.1. If using one recipient, then inoculate each well with 200 µL of 1x MC medium with recipient.
SECTION: Day of the transformation: Mixing donor and recipient.
11.2. If using many unique recipients, then use a pipette tip to inoculate each well with one colony.
SECTION: Day of the transformation: Mixing donor and recipient.
13. Shortly before 4 hours have elapsed, prepare the donor for transformation:

Is it necessary to filter the donor? (see Before Start)
SECTION: Day of the transformation: Mixing donor and recipient.
14. Is the donor growing in test tube or 96-well plate format?
SECTION: Day of the transformation: Mixing donor and recipient.
15. Aliquot 1 mL sample of the donor to a 1.7-microfuge tube.
SECTION: Day of the transformation: Mixing donor and recipient.
16. Spin the sample at 21,000 x g (max speed) in a centrifuge for 1 minute.

21000 x g, 1 min 

SECTION: Day of the transformation: Mixing donor and recipient.
17. Remove supernatant to a new microfuge tube. Take care not to disrupt cells and cell debris in pellet.
SECTION: Day of the transformation: Mixing donor and recipient.
18. Filter the supernatant through a 0.22 µm PES filter.

This filtrate is the source of donor DNA.
Note: cellulose acetate and nylon filters are equally effective, but PES is usually most cost effective.
SECTION: Day of the transformation: Mixing donor and recipient.
19. Donor DNA source may sit at room temperature while preparing the transformation tubes, but it can be stored at -30 °C.

SECTION: Day of the transformation: Mixing donor and recipient.
14. Aspirate directly from the donor culture and use this as the source of donor DNA.

Note: This aspiration of the donor can happen from either a test tubes or a 96-well plate.
Note: Due to the selective pressure, no spin is necessary.
SECTION: Day of the transformation: Mixing donor and recipient.
15. Filter away from the donor bacteria with a 0.22 µm filter type.

This filtrate is the source of donor DNA.
SECTION: Day of the transformation: Mixing donor and recipient.
16. Donor DNA source may sit at room temperature while preparing the transformation tubes, but it can be stored at -30 °C.
SECTION: Day of the transformation: Mixing donor and recipient.
20. Transfer 200 µL of the recipient to a sterile, 13mm glass tube (this is the transformation tube).

SECTION: Day of the transformation: Mixing donor and recipient.
21. Transfer 10 µL of donor filtrate to the transformation tube.

SECTION: Day of the transformation: Mixing donor and recipient.
16. Transfer 200 µL of the recipient to a sterile, 13mm glass tube (this is the transformation tube).

SECTION: Day of the transformation: Mixing donor and recipient.
17. Transfer 10 µL of donor lysate to the transformation tube.

SECTION: Day of the transformation: Mixing donor and recipient.
15. Is the donor growing in test tube or 96-well plate format?
SECTION: Day of the transformation: Mixing donor and recipient.
14. Aspirate directly from the donor culture and use this as the source of donor DNA.

Note: This aspiration of the donor can happen from either a test tubes or a 96-well plate.
Note: Due to the selective pressure, no spin is necessary.
SECTION: Day of the transformation: Mixing donor and recipient.
16. Transfer 200 µL of the recipient to a fresh well of a 96-well plate.
SECTION: Day of the transformation: Mixing donor and recipient.
17. Transfer 10 µL of donor lysate to the transformation well.
SECTION: Day of the transformation: Mixing donor and recipient.
17. Transfer 200 µL of the recipient to a fresh well of a 96-well plate.
SECTION: Day of the transformation: Mixing donor and recipient.
18. Transfer 10 µL of donor filtrate to the transformation well.
SECTION: Day of the transformation: Mixing donor and recipient.
18. Place transformations at 37 °C for 2 hours with aeration.
Use the same aeration methods as used for the donor cultures.

  

120 min 
SECTION: Day of the transformation: Mixing donor and recipient.
19. Dilute transformation cultures 1:10 and 1:100.
SECTION: Day of the transformation: Mixing donor and recipient.
21. Use 4-5 beads per petri plate to spread the cells evenly.

Note: do not allow the liquid to sit on the agar plate for too long before spreading.
SECTION: Day of the transformation: Mixing donor and recipient.
22. Incubate plates at 37 °C overnight, 30 °C for two days, or Room temperature for three days.

Note: the resulting colonies should be the desired transformants.
SECTION: Day of the transformation: Mixing donor and recipient.
20. Dispense 100 µL to 200 µL on appropriate selective plates.

Note: ensure the agar plates are not so dry that they prevent even spreading.
SECTION: Day of the transformation: Mixing donor and recipient.
18. Place transformations at 37 °C for 2 hours with aeration.
Use the same aeration methods as used for the donor cultures.

  

Note: Use the large 18 mm tubes with small caps or 1 mL pipette tips inside to hold the small 13 mm glass tubes (See Figure 1 in Loyo & Burton 2018, link in References Section).

120 min 

SECTION: Day of the transformation: Mixing donor and recipient.
19. Dilute transformation cultures 1:10 and 1:100.
SECTION: Day of the transformation: Mixing donor and recipient.
20. Dispense 100 µL to 200 µL on appropriate selective plates.

Note: ensure the agar plates are not so dry that they prevent even spreading.
SECTION: Day of the transformation: Mixing donor and recipient.
21. Use 4-5 beads per petri plate to spread the cells evenly.

Note: do not allow the liquid to sit on the agar plate for too long before spreading.
SECTION: Day of the transformation: Mixing donor and recipient.
22. Incubate plates at 37 °C overnight, 30 °C for two days, or Room temperature for three days.

Note: the resulting colonies should be the desired transformants.
SECTION: Day of the transformation: Mixing donor and recipient.
22. Place transformations at 37 °C for 2 hours with aeration.
Use the same aeration methods as used for the donor cultures.

  

Note: Use the large 18 mm tubes with small caps or 1 mL pipette tips inside to hold the small 13 mm glass tubes (See Figure 1 in Loyo & Burton 2018, link in References Section).

120 min 
SECTION: Day of the transformation: Mixing donor and recipient.
23. Dilute transformation cultures 1:10 and 1:100.
SECTION: Day of the transformation: Mixing donor and recipient.
24. Dispense 100 µL to 200 µL on appropriate selective plates.

Note: ensure the agar plates are not so dry that they prevent even spreading.
SECTION: Day of the transformation: Mixing donor and recipient.
25. Use 4-5 beads per petri plate to spread the cells evenly.

Note: do not allow the liquid to sit on the agar plate for too long before spreading.
SECTION: Day of the transformation: Mixing donor and recipient.
26. Incubate plates at 37 °C overnight, 30 °C for two days, or Room temperature for three days.

Note: the resulting colonies should be the desired transformants.
SECTION: Day of the transformation: Mixing donor and recipient.
19. Place transformations at 37 °C for 2 hours with aeration.
Use the same aeration methods as used for the donor cultures.

  

120 min 
SECTION: Day of the transformation: Mixing donor and recipient.
20. Dilute transformation cultures 1:10 and 1:100.
SECTION: Day of the transformation: Mixing donor and recipient.
21. Dispense 100 µL to 200 µL on appropriate selective plates.

Note: ensure the agar plates are not so dry that they prevent even spreading.
SECTION: Day of the transformation: Mixing donor and recipient.
22. Use 4-5 beads per petri plate to spread the cells evenly.

Note: do not allow the liquid to sit on the agar plate for too long before spreading.
SECTION: Day of the transformation: Mixing donor and recipient.
23. Incubate plates at 37 °C overnight, 30 °C for two days, or Room temperature for three days.

Note: the resulting colonies should be the desired transformants.
SECTION: Day of the transformation: Mixing donor and recipient.
20. In wells that contain 200 µL of the recipient, add 10 µL of donor filtrate to the transformation well. 
SECTION: Day of the transformation: Mixing donor and recipient.
21. Place transformations at 37 °C for 2 hours with aeration.
Use the same aeration methods as used for the donor cultures.

  

120 min 
SECTION: Day of the transformation: Mixing donor and recipient.
22. Dilute transformation cultures 1:10 and 1:100.
SECTION: Day of the transformation: Mixing donor and recipient.
23. Dispense 100 µL to 200 µL on appropriate selective plates.

Note: ensure the agar plates are not so dry that they prevent even spreading.
SECTION: Day of the transformation: Mixing donor and recipient.
24. Use 4-5 beads per petri plate to spread the cells evenly.

Note: do not allow the liquid to sit on the agar plate for too long before spreading.
SECTION: Day of the transformation: Mixing donor and recipient.
25. Incubate plates at 37 °C overnight, 30 °C for two days, or Room temperature for three days.

Note: the resulting colonies should be the desired transformants.
SECTION: Day of the transformation: Mixing donor and recipient.
17. In wells that contain 200 µL of the recipient, add 10 µL of donor filtrate to the transformation well.
SECTION: Day of the transformation: Mixing donor and recipient.
18. Place transformations at 37 °C for 2 hours with aeration.
Use the same aeration methods as used for the donor cultures.

  

120 min 
SECTION: Day of the transformation: Mixing donor and recipient.
19. Dilute transformation cultures 1:10 and 1:100.
SECTION: Day of the transformation: Mixing donor and recipient.
20. Dispense 100 µL to 200 µL on appropriate selective plates.

Note: ensure the agar plates are not so dry that they prevent even spreading.
SECTION: Day of the transformation: Mixing donor and recipient.
21. Use 4-5 beads per petri plate to spread the cells evenly.

Note: do not allow the liquid to sit on the agar plate for too long before spreading.
SECTION: Day of the transformation: Mixing donor and recipient.
22. Incubate plates at 37 °C overnight, 30 °C for two days, or Room temperature for three days.

Note: the resulting colonies should be the desired transformants.
SECTION: Day of the transformation: Mixing donor and recipient.
15. Is the donor growing in test tube or 96-well plate format?
SECTION: Day of the transformation: Mixing donor and recipient.
16. In wells that contain 200 µL of the recipient, add 10 µL of donor lysate to the transformation well.
SECTION: Day of the transformation: Mixing donor and recipient.
17. Place transformations at 37 °C for 2 hours with aeration.
Use the same aeration methods as used for the donor cultures.

  

120 min 
SECTION: Day of the transformation: Mixing donor and recipient.
18. Dilute transformation cultures 1:10 and 1:100.
SECTION: Day of the transformation: Mixing donor and recipient.
19. Dispense 100 µL to 200 µL on appropriate selective plates.

Note: ensure the agar plates are not so dry that they prevent even spreading.
SECTION: Day of the transformation: Mixing donor and recipient.
20. Use 4-5 beads per petri plate to spread the cells evenly.

Note: do not allow the liquid to sit on the agar plate for too long before spreading.
SECTION: Day of the transformation: Mixing donor and recipient.
21. Incubate plates at 37 °C overnight, 30 °C for two days, or Room temperature for three days.

Note: the resulting colonies should be the desired transformants.
SECTION: Day of the transformation: Mixing donor and recipient.
16. In wells that contain 200 µL of the recipient, add 10 µL of donor lysate to the transformation well. 
SECTION: Day of the transformation: Mixing donor and recipient.
17. Place transformations at 37 °C for 2 hours with aeration.
Use the same aeration methods as used for the donor cultures.

  

120 min 
SECTION: Day of the transformation: Mixing donor and recipient.
18. Dilute transformation cultures 1:10 and 1:100.
SECTION: Day of the transformation: Mixing donor and recipient.
19. Dispense 100 µL to 200 µL on appropriate selective plates.

Note: ensure the agar plates are not so dry that they prevent even spreading.
SECTION: Day of the transformation: Mixing donor and recipient.
20. Use 4-5 beads per petri plate to spread the cells evenly.

Note: do not allow the liquid to sit on the agar plate for too long before spreading.
SECTION: Day of the transformation: Mixing donor and recipient.
21. Incubate plates at 37 °C overnight, 30 °C for two days, or Room temperature for three days.

Note: the resulting colonies should be the desired transformants.
SECTION: Day of the transformation: Mixing donor and recipient.
9.2. Table for making 1 mL or 400 µL of 1x MC medium.
 

SECTION: Day of the transformation: Mixing donor and recipient.
9.1. Alternatively, standard 96-well plates may be used with 200 µL of 1x MC medium per well or square deep-well, 96-well plates may be used with 400 µL of 1x MC medium per well.

Note: ensure there is enough recipient culture for the number of unique transformations.