• Materials
  • For 500ml media:
  • 10g glucose (2%w/v)
  • 2.5g yeast extract (0.5% w/v)
  • 2.5g peptone (0.5% w/v)
  • 1.35g Na2HPO4 (0.27% w/v)
  • 0.75g citric acid (0.15% w/v)
  • 500ml distilled H20
  • 0.5mL cellulase if HS+cellulase media required
  • 7.5g of agar (agar-agar) if making HS-agar plates
  • (antibiotics as necessary)

1. Add 250ml dH20 to glucose in one bottle and 250ml dH20 to the rest in a second bottle. In incompletely distilled water, glucose will form a solid mass, so stir vigorously immediately after adding water. Autoclave both bottles to sterilize media and pour glucose solution in sterile conditions (next to a Bunsen burner or in a flow hood) into the second bottle. Autoclaving glucose separately from amino acids avoids Maillard reaction, which can result in the formation of toxic byproducts in the media.
2. Streak/inoculate Gluconacetobacter onto plates or into media.
3. Incubate plates at 30°C inverted. Colonies will appear in 48-72 hours.
4. Incubate liquid HS-cultures at 30 °C standing. Standing culturing results in low growth rate, but avoids the formation of cellulose non-producing mutants, which have been reported to appear in shaking conditions.
5. For quick growth, grow with shaking at 180rpm, 30C, with the addition of cellulase. NB! This selects for cellulose non-producing mutants. They can be identified as smooth colonies on plates (cellulose producing colonies have a rough colony morphology - see section Gluconacetobacter for images).

• Materials
  • HS+cellulase media
  • Ice bucket and ice
  • Temperature controlled centrifuge
  • 1mM HEPES (ph7.0) 80mL
  • 15% glycerol
  • 50 mL tubes
  • P1000 pipette and tips
  • Stripettes and automatic pipette for larger volumes
  • Shaker at 30 °C, 180rpm

1. If the goal of transformation is to produce cellulose-producing transformed G.xylinus, use regular HS media for culturing. If cellulose production after transformation is not primary and speed is required, HS-cellulase medium can be used. Usage of HS-cellulase medium during growth results in the formation of a higher number of cellulose negative mutants, but results in much higher transformation efficiencies, and requires less time. Even with HS-cellulose medium, cellulose producing colonies can be identified on the plate after transformation, as cellulose-producing colonies differ in morphology from cellulose non-producing colonies (see section Gluconacetobacter for images of colony morphology).
2. Inoculate 5ml of HS or HS+cellulase medium with Gluconacetobacter.
3. Incubate at 30°C, 180 rpm shaking overnight.
4. Next day, pour 30mL of HS or 15ml of HS+cellulase medium into each of four 50mL tubes. If using regular HS medium, vortex the tubes for 3 minutes to release cells from the cellulose pellicle
5. To each tube, add 1mL of overnight culture and incubate with shaking at 180rpm, 30C.
6. Incubate overnight or until OD600 of around 0.4-0.7 is reached. When using HS-cellulase media, OD600 can reach up to 0.6 -0.7, however when using regular HS medium, OD600 measurement is disturbed by the cellulose pellicle, and can reach up to 0.2. Vortex tubes for 3 minutes before taking the measurements.
7. If using regular HS medium, add 62ul (0.2% v/v) Celluclast cellulase to each tube and incubate at 30C, 180rpm shaking for 2 hours, or until the cellulose pellicle is degraded to completely release the cells. This does not result in higher formation of cel- mutants, as short incubation time and nutrient depletion does not allow for proliferation of cel- mutants.
8. Before continuing, set up the necessary materials:
   1. Pre-cool centrifuge to 4°C
   2. Prepare ice bucket
   3. Chill 1mM HEPES buffer and 15% glycerol buffer on ice
9. Once the cultures reach desired OD600, take them out of incubation and put them on ice for 10 minutes (the tube should feel cool).
10. From here on, keep cells always cool, at or below 4°C
11. After cooling, spin the tubes in a refrigerated (4°C) centrifuge for 12min at 4100rpm.
12. Pour off supernatant carefully, taking care not to pour off the pellet. G. xylinus does not pellet as easily as E.coli, most likely due to the buffering effects of cellulose. If the pellet is not attached to the wall after centrifugation, smear the pellet onto the wall of the tube and centrifuge again using longer centrifugation times. Re-suspend bacteria in 10mL HEPES: re-suspend first using 1ml HEPES and a P1000 pipette, then add 9ml of HEPES using a stripette; it is much easier to re-suspend the pellet fully using a P1000. Do not use a vortexer.
13. Pool the samples into a single 50ml tube.
14. Centrifuge again for 14 minutes at 4100 rpm and 4 °C temp. Use a balancer tube with 40ml water.
15. Pour off supernatant again, re-suspend pellet in 10mL ice-cold HEPES on ice as before.
16. Centrifuge again for 14 minutes at 4100 rpm, 4 °C.
17. Pour off supernatant and re-suspend pellet in 4mL ice cold 15% glycerol solution.
18. Pipette 50ul aliquots into tubes. Label tubes properly.
19. Store samples on ice for immediate use or freeze 50ul aliquots in-80°C. According to some reports, the efficiency of electrocompetent cells reduces after each freezing, so immediate use may result in highest efficiencies.

• Prepare the following
  • Plasmid DNA
  • Electrocompetent G.xylinus cells (in 50µl or 100µl aliquots)
  • Ice bucket and ice
  • 1mm path electrocuvettes
  • 1.5ml microcentrifuge tubes or PCR tubes
  • An electroporator - set at 2.5kV and 5.9ms
  • HS+cellulase medium
  • HS agar plates with appropriate antibiotic
  • One aliquot of electrocompetent cells for positive control and one aliquot for negative control.
  • If a plasmid known to work in G. xylinus exists, use this as positive control .

1. Set up the electroporator with correct settings: at 2.5kV, 6-8ms, 400ohm resistance, 25microF capacitance.
2. Prepare 800µL HS+cellulase media containing 1.5ml culture tubes and pre-heat to 30 °C. Prepare SOC medium at 37°C. Prepare a space for shaking.
3. Prepare ice bucket, place plasmid DNA and electrocuvettes on ice and thaw electrocompetent cells on ice. NB! Make sure DNA is desalinated before use – ionic solution can otherwise cause arcing.
4. Add 2 µL of plasmid DNA to 100 µL of concentrated cells in a cold microcentrifuge or PCR tube and mix well by pipetting. Add plasmid DNA also to one aliquot of electrocompetent E.coli cells (positive control; alternatively also add another known plasmid as pos. control). NB! Do not add plasmid DNA to one aliquot of electrocompetent G. xylinus (negative control)
5. Transfer the cell/DNA mixture and positive and negative controls to a cold 0.2-cm electroporation cuvette. Dry any water condensate outside of the cuvette (using labcoat), place the cuvette into the electroporator, and apply the pulse.
6. Transfer the pulsed cells into 800 µL of HS+cellulase medium in a culture tube. Transfer E. coli into SOC medium. NB! Prepare everything beforehand and do it quickly.
7. Incubate the culture tubes with shaking (170 rpm) at 30 °C overnight

During this step genomic DNA is tagmented (tagged and fragmented) by the Nextera transposome. The Nextera transposome simultaneously fragments the genomic DNA and adds adapter sequences to the ends, allowing amplification by PCR in subsequent steps.

Preparation

1. Prepare 200ul PCR tubes, ice bucket, ice
2. Remove the TD, TDE1, and genomic DNA from -15° to -25°C storage and thaw on ice.
3. In preparation for the next step of the protocol, ensure ST buffer is at room temperature, and there are no particles or precipitate visible in the solution.
4. Remove RSB from -15° to -25C storage and thaw at room temperature.
5. After thawing, ensure all reagents are adequately mixed by gently inverting the tubes 3–5 times, followed by a brief spin in a microcentrifuge. NOTE Ensure the reaction is assembled in the order described for optimal kit performance. The reaction does not need to be assembled on ice.
6. Label the PCR tubes with a smudge resistant pen.
7. Add 20 μl of genomic DNA at 2.5 ng/μl (50 ng total) to each PCR tube
8. ATCC: 3.27 of DNA, 16.7 of H2O
9. KI: 8.3ul of DNA, 11.7ul of H20
10. Add 25 μl of TD Buffer to the wells containing genomic DNA. Change tips between samples. NOTE:Calculate the total volume of TD for all reactions, and divide among an appropriate number of tubes in an 8-well PCR strip tube. Use a multichannel pipette to dispense into the NSP1 plate.
11. Add 5 μl of TDE1 to the tubes containing genomic DNA
12. Gently pipette up and down 10 times to mix the reaction.
13. Centrifuge at 280g for 1 minute
14. Place the PCR tubes into the thermocycler and run the following program:
    1. Heated lid on
    2. 55°C for 5 minutes
    3. Hold at 10°C
15. While tagmentation is ongoing, label 1.5ml tubes accordingly, and add 180ul of Zymo DNA binding buffer
16. Also, remove NPM, PPC, and the index primers from -15° to -25°C storage and thaw on a bench at room temperature.
17. Allow approximately 20 minutes to thaw NPM, PPC, and index primers
18. After tagmentation reaction has finished, proceed immediately to Zymo cleanup

Zymo cleanup protocol

1. Transfer 50 μl of tagmentation reaction into 1.5ml tubes containing Zymo binding buffer
2. Gently pipette up and down 10x to mix well
3. Transfer the solution to Zymo spin columns
4. Centrifuge at 10000rpm for 1 minute. (Increase speed for tubes if necessary, at around 10000rpm)
5. Discard the flow-through or the collecting tube and place the column into a new collection tube
6. Wash the Zymo spin column twice by:
   1. Pipette 300ul of Zymo wash buffer to the tubes
   2. Centrifuge at 10000rpm for 1 minute, discarding the flow through
   3. Repeat the wash step for a total number of 2 washes.
   4. Centrifuge at 10000rpm 1 min to ensure no wash buffer remains.
   5. Add 25 μl of RSB directly to the column matrix in each well
   6. Incubate the tubes for 5 minutes at room temperature.
   7. Centrifuge the tubes at 10000rpm at 20°C for 1 minute.
   8. Place the tubes on ice until proceeding to PCR

PCR

1. After NPM, PPC, and Index primers are completely thawed, gently invert each tube 3–5 times to mix and briefly centrifuge the tubes in a microcentrifuge. Use 1.7 ml Eppendorf tubes as adapters for the microcentrifuge.
2. Label PCR tubes according to your samples
3. Add 5 μl of index 2 primers (white caps) to each tube respectively.
4. Add 5 μl of index 1 primers (orange caps) to each tube respectively.
5. Add 15 μl of NPM (Nextera PCR master mix) to each well of the NAP1 plate containing index primers.
6. Add 5 μl PPC (PCR primer cocktail) to each well containing index primers and NPM.
7. Transfer 20 μl of purified tagmented DNA to the corresponding PCR tube
8. Gently pipette up and down 3–5 times to thoroughly combine the DNA with the PCR mix.
9. Change tips between samples to avoid index and sample cross contaminationy
10. Centrifuge at 280 xg at 20°C for 1 minute.
11. Perform PCR using the following program on a thermal cycler (NOTE: Ensure that the thermocycler lid is heated during the incubation.):
    1. 72°C for 3 minutes
    2. 98°C for 30 seconds
    3. 5 cycles of:
       1. 98°C for 10 seconds
       2. 63°C for 30 seconds
       3. 72°C for 3 minutes
    4. Hold at 10°C
12. SAFE STOPPING POINT – can store the DNA in thermocycler overnight, or at 2-8°C for 2 days

Clean-up of PCR using AMPure beads

1. Bring the AMPure XP beads to room temperature.
2. Prepare fresh 80% ethanol from absolute ethanol. (NOTE Always prepare fresh 80% ethanol for wash steps. Ethanol can absorb water from the air impacting your results.)
3. xCentrifuge the PCR tubes containing you’re the limited cycle PCR product at 280 xg for 1 min (20˚C) to collect condensation.
4. Label new 1.5ml tubes accordingly for purification steps
5. Transfer 50 μl of the PCR product from the PCR tubes into new 1.5ml tubes. Change tips between samples.
6. Vortex the AMPure XP beads for 30 seconds to ensure that the beads are evenly dispersed.
7. Add 30 μl of AMPure XP beads to each tube containing the PCR product. For 2x250 runs on the MiSeq, add 25 μl of AMPure XP beads to each tube.
8. Gently pipette mix up and down 10 times. (NOTE: Alternatively the solution can be mixed by shaking the tubes on a shaker at 1,800 rpm for 2 minutes.)
9. Incubate the tubes (containing AMPure beads and PCR product) at room temperature without shaking for 5 minutes.
10. Place the plate on a magnetic stand for 2 minutes or until the supernatant has cleared.
11. With the tubes on the magnetic stand, carefully remove and discard the supernatant. Change tips between samples. (NOTE If any beads are inadvertently aspirated into the tips, dispense the beads back to the plate and let the plate rest on the magnet for 2 minutes and confirm that the supernatant has cleared.)
12. With the tubes on the magnetic stand, wash the beads with freshly prepared 80% ethanol as follows:
    1. Add 200 μl of freshly prepared 80% ethanol to each sample well. You should not resuspend the beads at this time.
    2. Incubate the plate on the magnetic stand for 30 seconds or until the supernatant appears clear.
    3. Carefully remove and discard the supernatant.
13. With the tubes on the magnetic stand, perform a second ethanol wash as follows:
    1. Add 200 μl of freshly prepared 80% ethanol to each sample.
    2. Incubate the plate on the magnetic stand for 30 seconds or until the supernatant appears clear.
    3. Carefully remove and discard the supernatant. Use a P20 multichannel pipette with fine pipette tips to remove excess ethanol.
14. With the tubes still on the magnetic stand, allow the beads to air-dry for 15 minutes.
15. Remove the tubes from the magnetic stand. Add 32.5 μl of RSB to each well of the NAP2 plate.
16. Gently pipette mix up and down 10 times, changing tips after each column.(NOTE: Alternatively the solution can be mixed by shaking the tubes on a shaker at 1,800 rpm for 2 minutes.)
17. Incubate at room temperature for 2 minutes.
18. Place the tubes on the magnetic stand for 2 minutes or until the supernatant has cleared.
19. Label new 1.5ml tubes accordingly
20. Carefully transfer 30 μl of the supernatant into the new tubes. These will contain your purified library. Change tips between samples to avoid cross-contamination

• Materials per sample
  • 250ul P1 buffer (suspension buffer)
  • 250ul P2 buffer (Lysis buffer)
  • 350ul N3 buffer
  • 750ul PE buffer
  • 500ul PE buffer
  • Columns

1. Spin cells down at 4000rpm for 10 minutes
2. Discard supernatant (LB)
3. Resuspend pellet in P1 buffer
4. Transfer to labeled Eppendorf tube
5. Add P2 buffer. Solution should turn blue
6. Invert tubes 4-6 times, then wait for 2 minutes
7. Stop the reaction by adding N3 buffer and immediately inverting 4-6 times. Solution should turn clear
8. Centrifuge at 13000 rpm for 10 minutesv
9. Decant/pipette supernatant into mini-prep columns. Discard flow-through
10. Wash with PE buffer (750ul)
11. Centrifuge at 13000 rpm for 1 minutev
12. Discard flow-through. Add second wash of PE buffer (500ul)v
13. Centrifuge at 13000 rpm for 1 minute
14. Discard flow-through
15. Centrifuge empty columns at 13000 rpm for 1 minute to eliminate any excess wash buffer
16. Discard flow-through
17. Move columns into a labelled eppendorf
18. Add 30-40ul distilled water and wait for 2-3minutes
19. Elute DNA by centrifuging at 13000 rpm for 1 minute, do not discard flow-through. Discard column.
20. Nanodrop

• Materials
  • 2ul Cutsmart buffer (New England Biolabs)
  • 1ul Restriction enzymes
  • 12-16ul DNA to be digested
  • Distilled water (up to 20ul total volume)

1. Prepare mix
2. Incubate for 1-2h at 37 degrees

• Materials
  • 1g Agarose
  • 100mL 1X TAE buffer
  • 8uL SYBR Safe

1. Mix Agarose and 1x TAE buffer
2. Heat up until Agarose is dissolved
3. Add SYBR Safe
4. Pour into gel tray and let cool

• Materials
  • 1% Agarose gel DNA ladder
  • 6x loading dye
  • Electrophoresis cuvette

1. Set gel tray into cuvette, filled with 1x TAE buffer
2. Inoculate samples, previously dyed with 6x loading dye. Additionally, provided a DNA ladder for further reference of DNA sizes
3. Run gel at 110V for 30-40min

• Materials
  • 5mL Luria Broth
  • 5ul specific antibiotic
  • Loops (for colony picking or glycerol stock scraping)

1. Add Luria Broth into 50mL tube
2. Inoculate specific antibiotic
3. Scrape/pick glycerol stock surface/colony and transfer into falcon tube
4. Incubate at 37°C overnight