1. Assemble all reaction components in appropriately labelled PCR tubes
• See “DNA Template” below for details on different DNA templates
• Components common to all reactions can be combined and aliquoted individually (e.g. for reactions using the same primers but different templates, combine water + primers + Q5, aliquot into each tube, then add the different templates)
2. Collect all the liquid to the bottom of the tubes by tapping the bottom of the tubes on a hard surface a few times, or alternatively by centrifuging for a few seconds
3. Place the PCR tubes into a thermocycler and run the below program

DNA Template

• If using plasmid DNA as a template, miniprepping beforehand is advised for increased quality template, then use NanoDrop to determine concentration (although, colony PCR can also work)
• If using genomic DNA as a template, use colony PCR

Colony PCR

1. Pick up a small amount of a single colony with a pipette tip and resuspend in 10 µL sterile dH2O in an appropriately labelled PCR tube
2. Transfer 5 µL of the dH2O to 20 µL sterile 20 mM NaOH in an appropriately labelled 1.5 mL Eppendorf tube or PCR tube (leaving 5 µL for later use in culturing)
3. Heat the 25 µL solution to 95 °C for 15 minutes in a heat block (alternatively, you can put the PCR tubes in a thermocycler)
4. Centrifuge the boiled solution to form a pellet (~5 minutes at 13k RPM) and use 1 µL of supernatant as the DNA template
5. (Alternatively, a small amount of single colony can be added to 50 µL PBS, then use ~0.5 µL of this solution as the DNA template (less is generally better), then extend the initial denaturation step to 5 minutes — however, this is less consistent than the other approach and doesn’t save much time)

Based on the protocols from Bennett Lab, Rice University and University of Cambridge 2022 iGEM Team

Pouring a Gel

1. Add 1% agarose to 1x TAE (0.5 g agarose for a 50 mL gel, 1 g for a 100 mL gel; can increase/decrease agarose concentration for small or large fragments)
2. Microwave the mixture for ~1-2 minutes until the solution is clear
3. Let the solution cool, or cool it under a tap, until it is cool enough to handle
4. Add 1 µL of SYBR Safe stain per 100 mL (0.5 µL for a 50 mL gel, 1 µL for a 100 mL gel)
5. Pour the solution into a gel mould containing an appropriate comb and allow to set for at least 20-30 minutes

Running a Gel

1. Add 8 µL of ladder to the first well
2. Add 1 µL of 6x loading buffer to empty PCR tubes, then add 5 µL of sample into each tube, triturate to mix, then load 5 µL into each well
3. Run at 90V for ~45-60 minutes (45 minutes for 50 mL gel, 60 minutes for 100 mL gel)

Visualising a Gel

1. View under a UV or blue light transilluminator

PCR purify or gel purify the PCR product by following the instructions for the kit you are using. We recommend doing PCR purification if you only see a single band (of the expected size) during electrophoresis, but doing gel purification if you see multiple bands and need to isolate DNA from a single band

Note: NanoDrop should be performed with purified DNA, not unpurified PCR products etc.

1. Clean the NanoDrop by wiping clean with a lint-free cloth, load 2 µL of distilled water, closing the arm, waiting for 1 minute, then wiping clean again
2. Load 2 µL of distilled water, click “Blank”
3. Click “Measure” to confirm that there is no contaminating DNA (should be close to zero, +/- ~2ng/uL)
4. Wipe clean, load 2 µL of sample, click “Measure” (measure at least twice to confirm the reading is consistent, you should see a fairly sharp peak around 260 nm)
5. After you’re done, wipe clean, load 2 µL of distilled water, close the arm, wait for 1 minute, then wipe clean again, leave some folded clean tissue below the arm

1. Assemble all reaction components in appropriately labelled PCR tubes
• Components common to all reactions can be combined and aliquoted individually (e.g. for reactions using the same REs but different inserts, combine water + buffer+ ligase + REs, aliquot into each tube, then add the different inserts)
• Since the T4 ligase buffer contains ATP, aliquot the stock solution out to minimise the number of freeze-thaw cycles
2. Collect all the liquid to the bottom of the tubes by tapping the bottom of the tubes on a hard surface a few times, or alternatively by centrifuging for a few seconds
3. Place the PCR tubes into a thermocycler and run the below program (for assembly with a small number of fragments, you can reduce the time and number of digestion/ligation cycles)
4. (If the assembly contains an internal cut site for the used type IIS restriction enzyme, you can perform an additional final ligation step of 16 °C for ~20 minutes, assuming that the unwanted overhang made during the assembly doesn’t bind any of the other overhangs)

DNA Concentration Calculator

You can use this Golden Gate DNA Concentration Calculator to calculate the correct volume of each component to add to the reaction

Heat Shock Transformation

For NEB® 5-alpha competent E. coli, follow the protocol on NEB’s website

Electroporation

Preparing Competent Cells

1. Inoculate a single colony of E. coli into 2 mL LB containing the appropriate antibiotic. Incubate shaking at 37 °C overnight
2. Dilute overnight culture 1 mL into 25 mL LB containing appropriate antibiotic(s). Shake at 37 °C until OD600 = 0.6 - 0.8 (~1.5-3 hours).
3. Centrifuge at 5,000 RPM at 4 °C for 10 minutes. Resuspend pellet in 25 mL ice-cold 10% glycerol
4. Repeat step 3
5. Centrifuge at 5,000 RPM at 4 °C for 10 minutes. Resuspend pellet in 1 mL ice-cold 10% glycerol
6. Transfer into a 1.5 mL microcentrifuge tube.
7. Centrifuge at 14,000 RPM at 4 °C for 1 minute. Resuspend pellet in 250 µL ice-cold 10% glycerol.
8. Use immediately or make 50 µL aliquots in ice-cold 1.5 mL tubes and store at -80 °C

Electroporation Transformation

1. Add 1 µL plasmid DNA or ligation reaction to 50 µL of competent cells
2. Add the mixture to the side wall of an ice-cold electroporation cuvette
4. Place the electroporation cuvette into the shocking chamber
5. Press PULSE 2x (time constant should be 5.0-6.0)
6. Add 500 µL SOC medium to cuvette and transfer to a ~15 mL culture tube
7. Incubate shaking at 37 °C for 1 hour
8. Plate on selection plates
9. Incubate at 37 °C overnight

SOC recipe on Cold Spring Harbor Protocols

Start an overnight culture with the transformed E. coli, grow at 37°C for 12-18 hours, then follow the steps for the miniprep kit you are using (we recommend eluting in nuclease-free water)

1. In a 50 mL falcon tube, inoculate 5 mL of SPC media with a single colony of *B. subtilis*
2. Incubate shaking at 37 °C until the OD600 is constant (usually ~7 hours)
3. Dilute 10x into 10 mL warm SPII media (1 mL cells + 9 mL SPII) in a 50 ml falcon tube
4. Incubate shaking at 37 °C for 90 minutes
5. Centrifuge at 4,000 rpm for 3 mins
6. Resuspend the pellet in 1 mL of the supernatant (discard the rest of the supernatant)
7. If not transforming immediately, add 200 µL of sterile 50% glycerol. Mix well and make 100 µL aliquots of the competent to store at -80 °C (can be stored at -80 °C for ~1 year)
8. If the cells are going to be transformed on the same day, add 100ul of ME to 100ul of cell suspension. Add DNA (approx.. 1ug-2ug in a max volume of 10ul). Incubate the cells with the DNA in the shaker at 37 °C for 30-60 min. Plate in selection plates.
9. To transform, quickly thaw an aliquot of competent cells (or use freshly made competent cells), add 100ul of ME, add ~1-2 µg of DNA (in a max volume of 10 µL), then incubate shaking at 37 °C for ~60 minutes
10. Plate on selection plates
11. Incubate at 37 °C overnight

Make fresh and filter sterilise with a 0.2 µm filter

Filter sterilise with a 0.2 µm filter. Keep at room temperature for months. Discard if precipitate appears

1. Follow the above B. subtilis transformation protocol and plate on erythromycin plates (0.5 µg/mL)
2. Pick ~4 colonies from transformation plate and inoculate each colony in 3 mL of LB. Incubate shaking at 25°C for ~10 hours
3. Transfer 30 μL of each culture into 3 mL of fresh LB, then incubate at 25 °C overnight
4. Transfer 30 μL of each culture into 3 mL of fresh LB, then incubate at 25 °C for ~10 hours
5. Transfer 30 μL of each culture into 3 mL of fresh LB, then incubate at 25 °C overnight
6. Transfer 30 μL of each culture into 3 mL of fresh LB. Incubate at 37 °C for ~3 hours (until the OD600 of the cultures is ~1.1-1.3)
7. Serially dilute to ~10^-6, then spread 200 μL of each dilution on LB plates. Incubate at 37 °C overnight (can increase or decrease serial dilution to get a high number of single colonies)
8. Pick ~10 colonies from the LB plate and restreak on LB and LB + erythromycin (0.5 µg/mL) plates. Incubate at 37 °C overnight (restreak = pick the colony with a sterile pipette tip/toothpick and draw a line/squiggle on a section of an LB plate, then draw a line/squiggle on a section of an LB + erythromycin plate)
9. Successful transformants should be susceptible to erythromycin, so will grow on LB but will not grow on LB + erythromycin – perform colony PCR on these colonies to confirm the correct transformation

Based on the protocol from Wozniak and Simmons (2022)

1. Inoculate a single colony of B. subtilis in around ~5 mL of TSB and incubate at 30 °C overnight
2. Measure the OD600 of the cultures and dilute to OD600 = 0.02
3. Add 200 µL to each well of a tissue culture treated, flat-bottom 96-well plate (use 200 µL of sterile TSB for a negative control)
4. Seal the 96-well plate with an adhesive gas permeable seal (e.g. 4titude® moisture barrier seal)
5. Incubate at 30 °C for the desired amount of time (~72 hours for mature biofilm for B. subtilis)
6. Gently remove the media using a multichannel pipette and wash with 200 µL of PBS (gently add the PBS to each well, then gently aspirate and discard from each well)
7. Leave to air dry for 30 minutes
8. Add 150 µL of 0.25% safranin in 10% ethanol (“Gram’s safranin”) to each well, being carefully to avoid getting safranin on the sides on the wells
9. Incubate at room temperature for 10 minutes, then gently aspirate the safranin from each well with a multichannel pipette and wash ~10 times by pipetting and aspirating distilled water into each well, until the water is colourless following washing (an automated plate washer can also be used for this step, however it should not be used for other wash steps that require gentle aspiration)
10. Leave to air dry for 1 hour
11. Solubilise the remaining stain by adding 150 µL of 33% acetic acid to each well
12. Incubate at room temperature for 30 minutes, then transfer to a new 96-well plate using a multichannel pipette and measure absorbance at 530 nm using a plate reader

Adapted from Meyer et al. (2017)

1. Inoculate a single colony of B. subtilis in around ~5 mL of TSB and incubate at 30 °C overnight
2. Measure the OD600 of the cultures and dilute to OD600 = 0.02
3. Add 200 µL to each well of a tissue culture treated, flat-bottom 96-well plate (use 200 µL of sterile TSB for a no biofilm condition)
4. Seal the 96-well plate with an adhesive gas permeable seal (e.g. 4titude® moisture barrier seal)
5. Incubate at 30 °C for the desired amount of time (~72 hours for mature biofilm for B. subtilis)
6. Gently remove the media using a multichannel pipette, then gently add 200 µL of OD600 = 0.02 E. coli or Pseudomonas aeruginosa (or other invading organisms) in sterile TSB
7. Incubate at 30 °C for the desired amount of time
8. Gently remove the media using a multichannel pipette and wash twice with 200 µL of sterile PBS (gently add the PBS to each well, then gently aspirate and discard from each well)
9. Add 100 µL of sterile LB to each well
10. Using sterile P200 pipette tips, scratch/scrape the bottom of each well to remove adhered biofilm (sonication could also be used to this and would likely reduce variability, if you have access to this)
11. Transfer the 100 µL of LB to sterile PCR/microfuge tubes and vortex
12. Serially dilute in sterile LB and plate 6 µL of each serial dilution onto an LB plate and selective plate (we used LB + ampicillin with an E. coli strain containing ampicillin resistance; we plated 6 serial dilutions of 10^-3 to 10^-8 in a 6x6 grid on standard size LB plates)
13. Incubate at 30 °C overnight, then count the number of colonies on each plate

1. Inoculate a single colony of B. subtilis in 5 mL DSM. Incubate shaking at 37 °C until OD600 = 0.45-0.6 (usually a few hours)
2. Dilute 1 mL into 9 mL of warm DSM. Incubate shaking at 37 °C for 48 hrs
3. Transfer half of the culture to a sterile tube, then heat in a water bath at 70 °C for 20 minutes (to kill vegetative cells, but spores can survive)
4. Serially dilute the heated and unheated samples in sterile LB and plate 6 µL of each serial dilution onto an LB plate – the CFU count for the heated samples can be used as a readout for spore count, whilst the CFU count for unheated samples can be used as a readout for viable cell count and spore count