Team:Lambert GA/Experiments

EXPERIMENTS

CLONING PROTOCOLS

Restriction Digest

Reagent Amount
Restriction Enzyme 1 1µL
Restriction Enzyme 2 1µL
10x Cutsmart Buffer 5µL
Insert or Vector DNA 1µg
Nuclease Free Water / Sterile Water To 50µL
  1. Shave ice in styrofoam container
  2. Lable PCR tubes
  3. Add all reagents to tube
  4. Incubate for 30 minutes at 37°C
  5. Incubate for 20 minutes at 65°C

Ligation

Reagent Amount
T4 Ligase Buffer 2µL
Vector DNA 50ng
Insert DNA 150ng
Nuclease Free Water To 20ng
T4 DNA Ligase To 50µL
  1. Shave ice
  2. Label PCR tubes
  3. Add all of the reagents to your tube
  4. Incubate for 10 min at room temperature
  5. Incubate for 10 min at 65°C

Transformation

  1. Label tubes with initials, date, and what is being transformed
  2. Place all tubes on ice
  3. Put SOC/LB and antibiotic plates in the incubator to warm at 37°C
  4. Pipette 1ul of the desired plasmid(s) and 1ul of PUC19 into the corresponding labeled transformation tubes
  5. Thaw competent cells on ice for 10 minutes or just until the cells inside are moving when you flick the tube
  6. Pipette 50ul of competent cells into each labeled transformation tube on ice
  7. Incubate tubes on ice for 30 minutes
  8. Heat shock tubes at 42°C for 45 seconds
  9. Put on ice 2 minutes
  10. Add 950ul of SOC/LB media into each tube with cells
  11. Place tubes shaking in the incubator for 60 minutes at 37°C
  12. Pipette 120-150ul of cells with SOC/LB media onto warm plates with the correct antibiotics
  13. Place antibiotic plates in the incubator at 37°C for 24 hours to let cells grow

Inoculation for Cultures

  1. Prepare Liquid LB
  2. When ready to grow culture, add Liquid LB to a tube or flask and add your antibiotic that your cells are resistant to the correct concentration to create a 1 milliliter of LB to 1 microliter of antibiotic (typically use 5 ml: 5 ul)
  3. Using a sterile pipette tip select a single colony from your LB agar plate
  4. Drop the tip into liquid LB and antibiotic, then swirl
  5. Cover culture with sterile aluminum foil or cap that is not airtight
  6. Incubate culture at 37℃ for 12-18 hours in a shaking incubator
  7. After incubation, check for growth
  8. For long-term, storage continue with creating a glycerol stock

Miniprep

  1. Grow 1-5 mL culture overnight in a 10mL-20mL culture tube.
  2. Centrifuge at 2500 xg for 10 minutes at room temperature. Decant or aspirate and discard culture media.
  3. Add 250 uL of solution 1 mixed with RNase. Vortex to mix, then transfer suspension into a new 1.5 mL microcentrifuge tube.
  4. Add 250 uL of solution 2. Invert until there is clear lysate.
  5. Add 350 uL to solution 3, insert until white precipitate forms. Centrifuge at 13,000 xg for 10 minutes.
  6. Insert mini column into a 2 mL collection tube.
  7. Transfer the clear supernatant into a mini-column using a micropipette. Centrifuge at 13,000xg for 60 seconds. Discard filtrate and reuse the collection tube.
  8. Add 500 uL of HBC Wash Buffer diluted in ethanol. Centrifuge at 13,000xg for 60 seconds. Discard filtrate and reuse the collection tube.
  9. Add 700 uL of the DNA Wash Buffer diluted in ethanol. Centrifuge at 13,000xg for 30 seconds, Discard filtrate, and reuse collection tube.
  10. Centrifuge empty mini-column at 13,0000xg for 2 minutes to remove ethanol.
  11. Transfer mini-column to a nuclease-free 1.5 mL microcentrifuge tube.
  12. Add 50 uL of Elution Buffer. Let it sit at room temperature for 5 minutes. Centrifuge at 13,000xg for 60 seconds.<,li>
  13. Store eluted DNA at -20℃

Gel Electrophoresis

Making Gels:

  1. Mix 0.5g of agarose with 50ml of 1x TAE buffer in a 100-250ml Erlenmeyer flask
  2. Heat up the solution until it turns clear
  3. Let the solution cool down until it reaches 65°C
  4. Once the solution measures 65°C, add 6ul of SybrSafe in and gently shake the flask to mix the solution
  5. Pour the solution into a gel chamber and put in the gel comb
  6. Wait for the gel to solidify before loading

Running the Gel

  1. Once solidified, fix the gel’s position so that the wells of the gel are at the end of the chamber and DNA runs to red
  2. Pour used 1x TAE buffer into the gel chamber evenly to completely cover the gel
  3. On a piece of parafilm, mix 5ul of DNA and 1ul of purple loading dye using a micropipette
  4. Add the mixed 6ul solution into each well
  5. Add 6ul of 2-log ladder into an empty well
  6. Connect the electrodes by closing the gel chamber and connecting the to the power supply
  7. Set power supply for 90 volts and 45 minutes
  8. Turn on the power supply and make sure bubbles are rising on the sides of the gel chamber
  9. Once gel electrophoresis is completed, put the gel under UV (ultraviolet) light to compare the bands of DNA to the bands of the ladder

PCR

Reagent Amount
Sterile Water To 25 uL (normally 9uL)
Q5 High Fidelity Master Mix 12.5 uL
10 uM diluted forward primer 1.25 uL
10 uM diluted reverse primer 1.25 uL
Template DNA 1uL
  1. Add reagents to labeled PCR tube
  2. Set thermocycler with new annealing temperature and elongation times (annealing time and temperature depends on your part to PCR)
  3. Run PCR

RPA

  1. Prepare a primer pre-master mix (per reaction) in the following order:
    Reagent Amount
    2x Reaction Buffer 25uL
    dNTPs 1.8 mM concentration 9.2uL
    10x Basic E-mix 5uL
    Primer A (Forward Primer) 2.4 uL
    Primer B (Reverse Primer) 2.4 uL
    Vortex and spin briefly
  2. Add 2.5 μl 20x Core Reaction Mix4 (per reaction) to tube lid. Mix by 10x full inversions and spin briefly. Master mix is now complete. Pipette mix before use.
  3. Add 46.5 μl3 master mix to 0.2 ml PCR tubes.
  4. Add 2.5 μl of 280mM MgOAc and 1 μl template to the tube lid. DNA and MgOAc should be kept separate in the tube lid prior to spin-down. Spin in MgOAc/template, mix well (6x inversions) to start reaction. Spin briefly. Warning: RPA reactions start as soon as MgOAc is added!
  5. Incubate at 37-42°C for 20-40 minutes. For low template copies, remove strip after 4 mins, mix by 6x full inversions and spin briefly, replace in heating device.
  6. After step 5, clean amplicons before running on an agarose gel

Colony PCR

Reagent Amount
Q5 High-Fidelity 2X Master Mix 12.5uL
10uM Forward Primer VF2 1.25 uL
10uM Reverse Primer VR 1.25 uL
10 uM diluted reverse primer 1.25 uL
Nuclease-Free Water To 25uL
  1. Choose colony and dilute with 40uL of water
  2. Take 1uL out for the colony PCR
  3. Put all together in PCR tube
  4. Place in Thermocycler

PCR Cleanup

  1. Add 1:1 volume of binding buffer to completed PCR mixture
  2. Transfer up to 800 uL to GeneJET purification column
  3. Centrifuge for 60 seconds and discard flow through
  4. Add 700 uL wash buffer to the purification column
  5. Centrifuge for 60 seconds and discard flow through
  6. Centrifuge again for 60 seconds (dry spin) and discard flow through
  7. Transfer purification column to 1.5 uL microcentrifuge tube
  8. Add 30 uL elution buffer
  9. Wait 5 minutes
  10. Centrifuge for 1 minutes
  11. Store at -20 degrees C

CELL-FREE PROTOCOLS

Dr. Adam Silverman from Jewett Lab at Northwestern University shared his published lysate preparation and reagent preparation protocol from Supplementary Information for: “Deconstructing cell-free extract preparation for in vitro activation of transcriptional genetic circuitry” (Silverman, Kelley-Loughnane, Lucks, & Jewett, 2018). Lambert_GA made minor changes specific to our equipment and experiments.

Lysate Preparation [1]

Day 1: (5 minutes)

  1. Streak a fresh plate of the BL21 (DE3) cells onto a fresh plate (For Nar testing use cells with NarX membrane-bound proteins expressed.

    2. Day 2: (1 hour)

  2. Prepare and autoclave one liter of 2X YT + P media (16 g tryptone, 10 g yeast extract, 5 g sodium chloride, 7 g potassium phosphate dibasic, 3 g potassium phosphate monobasic, and water to one liter).
  3. Prepare and autoclave 200 mL of wash buffer (50 mM Tris base, 14 mM magnesium glutamate, 60 mM potassium glutamate) and pH to 7.7 using acetic acid.
  4. Prepare and autoclave 600 mL of dialysis buffer (5 mM Tris base, 14 mM magnesium glutamate, 60 mM potassium glutamate) and pH to 8.2 using Tris.
  5. Separately autoclave two 1L baffled culture flasks and lids, a stir bar, a spatula.
  6. Sixteen hours (for plain BL21) and twenty-four (NarX-enriched cells) before the beginning of the culture prep on Day 3, inoculate a 30 mL starter culture from the plate with antibiotic as appropriate into a 250 mL flask and grow overnight.

    7. Day 3: (12 hours, or 6 + 6 hours split across two days)

  7. Thirty minutes before inoculation, warm up the 2X YT+P to 37°C in an incubator.
  8. After the 16-hour (for plain BL21) and twenty-four (NarX-enriched cells) growth culture is complete, remove 20 mL of the 2X YT+P media to serve as the blank for measuring optical density. Then, inoculate the 1L growth culture with 20 mL of the saturated starter culture with antibiotic as appropriate.
  9. During the growth phase, chill all centrifuges, buffers, falcon tubes, and glassware to 4°C, and thaw 1.1 mL of 1M DTT. Grow to optical density (OD600) 3.0 ± 0.2 at 37°C and shaking at 200 rpm, which should require around 4 hours for BL21 (DE3) cells.
  10. Upon reaching the harvest optical density, immediately divide the cell culture into two centrifuge bottles on ice, balance, and centrifuge for 20 minutes at 3260XG (highest setting) at 4°C. From this point forward, keep the cells on ice at all times.
  11. During the first spin, add 400 μL of 1M DTT to the wash buffer so it is at a final concentration of 2 mM.
  12. Weigh and record the mass of two clean 50 mL Falcon tubes. Keep on ice.
  13. At the end of the spin, carefully decant the supernatant, taking care not to disturb the pellet. Using the spatula, transfer each pellet to a 50 mL Falcon tube, wiping the wet pellet on the side of the Falcon tube to maximize exposed surface area.
  14. Resuspend each cell pellet in 25 mL of wash buffer, vortexing each tube for 20 seconds at a time until the suspension is homogeneous and no solid is visible.
  15. Balance the Falcon tubes and centrifuge for 15 minutes at 3260XG at 4°C.
    16. vAt the end of the second spin, carefully decant the supernatant and resuspend each pellet in 25 mL of wash buffer, again vortexing in 20-second intervals. Once homogeneous, balance the two Falcon tubes and centrifuge for 15 minutes at 3260XG at 4°C.
  16. At the end of the third spin, carefully decant the supernatant and resuspend each pellet in 25 mL of wash buffer, again vortexing in 20-second intervals. Once homogeneous, balance the two Falcon tubes and centrifuge for 20 minutes at 73260XG at 4°C.
  17. At the end of the fourth spin, carefully decant the supernatant. Wipe all exposed surfaces of the tube and dry any residual liquid inside the tube. Weigh each pellet. If harvested around OD 3.0, each pellet should weigh around 1.5-3 g.
  18. Optional: At this point, the cells can be flash-frozen in liquid nitrogen and stored at -80°C. The subsequent steps take around 5 hours and should be done in the same day. After flash freezing, allow the cells to thaw for 1 hour on ice before attempting the next step.
  19. Resuspend each pellet in 1 mL wash buffer per gram of cell pellet by vortexing.
  20. Let the suspensions rest for 5-10 minutes. Chill a tabletop centrifuge to 4°C prior to the next step.
  21. Aliquot out 1.4 mL of the suspension into four total 1.7 mL Eppendorf tubes (total of 5-6 mL suspension from the two pellets).
  22. Sonicate the suspensions on ice. The optimal sonication energy may be strain-specific and require some tuning. The lysed suspensions should turn brown and become much less viscous.
  23. Add 4 μL of 1 M DTT to each 1.7 mL tube after lysis.
  24. After lysing all four tubes, centrifuge for 10 minutes at 12,000XG and 4°C.
  25. If the lysis has gone well, there will be a separation of the clarified lysate into three bands--a clear top band with milkier opaque cell debris at the bottom and an intermediate band between. Pipette off the top supernatants (approximately 800 μL per tube from 1.4 mL lysate) and combine into two fresh Eppendorf tubes.
  26. Cover the tubes with aluminum foil and incubate shaking at 37°C and 200 rpm for 80 minutes for the runoff reaction.
  27. After the runoff reaction is complete, centrifuge the tubes at 12,000XG for 10 minutes at 4°C. The tubes will go opaque again during the runoff reaction, and this centrifugation step will remove any additional protein translated during the incubation.
  28. Add 600 μL of 1M DTT to the dialysis buffer to a final concentration of 1 mM and transfer to the beaker with a stir bar. Soak a 10K MWCO dialysis cassette in the buffer for at least five minutes. Dry the cassette without blotting the membrane.
  29. After the centrifugation is complete, pipette off the supernatant, taking care not to remove any pellet.
  30. Load the dialysis cassette with this clarified extract. If no extract has yet been lost, this should be around 2-3 mL and will be faintly yellow but transparent.
  31. Dialyze with no buffer exchanges for three hours at 4°C.
  32. After the dialysis is complete, remove the extract from the cassette into two fresh 1.7 mL tubes and do a final spin at 12,000XG for 10 minutes at 4°C. There should be a very small pellet. Transfer the supernatant from this spin into a fresh tube--this is the final extract. Aliquot into smaller tubes at 35 μL each and flash-freeze on liquid nitrogen. From a one-liter culture prep, the yield should be around 2.5 mL total, or about 70 tubes containing 35 μL. Store at -80°C until use.

Cell-Free Reagent Preparation [1]

The cell-free reaction is composed of three major constituents: the extract (approximately 30% by volume; refer to Supplemental; Extract Preparation Protocol for detailed preparation notes); the Reaction Buffer (approximately 30% by volume); and the mixture of DNA and inducers supplied to each individual reaction (the balance, approximately 40% and made up to the extra volume with water). The reaction buffer itself has five major constituents which are maintained in separate stocks and mixed fresh for every reaction:

Salt Solution Stock (23.2% of Reaction Buffer by volume):

  • 15X* mM magnesium glutamate (Sigma 49605; stock solution prepared in water to 500 mM)
  • 150 mM ammonium glutamate (MP Biomedicals 02180595; stock solution prepared in water to 2.8M)
  • 1950 mM potassium glutamate (Sigma G1501; stock solution prepared in water to 3.5M)

*The magnesium concentration of the salt solution stock should be prepared to 15X the desired final reaction concentration. For example, if the final concentration of Mg in the reaction is set to be 16 mM Mg, then the salt solution stock should be at 15X16 = 240 mM. We recommend preparing a range of salt solutions and optimizing reaction conditions for each new extract or cell-free construct, as magnesium has an especially strong impact on the reaction yield.

NTP Master Mix Stock (23.2% of Reaction Buffer by volume):

Prepare to a final stock concentration:

  • 18 mM ATP (Sigma A-2383; prepared from an approximately 0.5M stock solution in water and pHed with 10N KOH)*
  • 12.75 mM GTP (Sigma G-8877; prepared from an approximately 0.5M stock solution in water and pHed with 10N KOH)*
  • 12.75 mM UTP (Sigma U-6625; prepared from an approximately 0.5M stock solution in water and pHed with 10N KOH)*
  • 12.75 mM CTP (Sigma C-1506; prepared from an approximately 0.5M stock solution in water and pHed with 10N KOH)*
  • 0.51 mg/mL folinic acid (Sigma 47612; prepared from a 9 mg/mL stock solution in water)
  • 2.559 mg/mL E. coli Mre 600 tRNA (Roche 10109541001; prepared from a 50 mg/mL stock solution in water)

*Each nucleotide stock concentration is validated using a NanoDrop and the following molar extinction coefficients:

  • ATP: 15.4 mM-1 cm-1 at 259 nm
  • GTP: 13.7 mM-1 cm-1 at 253 nm
  • UTP: 10.0 mM-1 cm-1 at 262 nm
  • CTP: 9.0 mM-1 cm-1 at 271 nm

Reagent Mix Stock (27.8% of Reaction Buffer by volume):

  • 372 mM HEPES (Sigma H3375, from a 1M, pH 7.4 stock solution prepared in water brought to pH with 10N KOH, and filter-sterilized)
  • 9.8 mM spermidine (Sigma S2626, from a 250 mM stock solution prepared in water)
  • 6.6 mM putrescine (Sigma P5780, from a 250 mM stock solution prepared in water)
  • 26 mM oxalic acid (Sigma P0963, from a 1M stock solution prepared in water)
  • 1.76 mM CoA (Sigma C3144, from a 50 mM stock solution prepared in water)
  • 2.61 mM NAD (Sigma N8535, from a 100 mM stock solution prepared in water) from a 100 mM stock solution prepared in water)

The desired concentrations are obtained by mixing 860 μL of 1M HEPES, 90 μL of 250 mM spermidine, 60 μL of 250 mM putrescine, 60 μL of 1M oxalic acid, 80 μL of 50 mM CoA, and 60 μL of 100 mM NAD.

Amino Acid Stock (13.9% of Reaction Buffer by volume):

Amino acids are prepared from separate powders to a total final concentration in the stock of 50 mM of each amino acid. The following instructions are for preparing 40 mL of this stock solution:

  1. Add 0.234 g L-valine (Sigma V0500), 0.408 g L-tryptophan (Sigma T0254), 0.33 g L-phenylalanine (Sigma P2126), and 0.262 g L-isoleucine (Sigma I2752) to 25 mL sterile water. Shake 10 minutes at 37oC or until soluble.
  2. Add 0.262 g L-leucine (Sigma L8000) and 0.242 g L-cysteine (Sigma C7352). Shake 10 minutes at 37oC or until soluble.
  3. Add 0.298 g L-methionine (Sigma M9625), 0.178 g L-alanine (Sigma A7627), 0.348 g L-arginine (Sigma A8094), 0.264 g L-asparagine (Sigma A0884), 0.266 g L-aspartic acid (Sigma A8949), 0.406 g L-glutamic acid, potassium salt (Sigma G1501), 0.150 g L-glycine (Sigma G8898), and 0.292 g L-glutamine (Sigma G3126). Shake 10 minutes at 37oC or until soluble.
  4. Add 0.308 g L-histidine (Sigma H8000), 0.365 L-lysine (Sigma L5501), 0.230 g L-proline (Sigma P0380), 0.210 g L-serine (Sigma S4500), 0.238 g L-threonine (Sigma T8625), and 0.362 g L-tyrosine (Sigma T3754).
  5. Add water to a final volume of 40 mL. Amino acids will not go into solutions after the addition of tyrosine, so vortex frequently to measure.
  6. Shake for 15 minutes in a 37oC incubator.
  7. Flash freeze the amino acid solution.

Energy Mix Stock (11.6% of Reaction Buffer by volume):

  • Prepare 1M PEP (phosphoenolpyruvate monopotassium salt; Roche 10108294; prepared to 1M at pH 7 using 10N KOH).

The final buffer mix is then prepared at a ratio of 1 : 1 : 1.2 : 0.6 : 0.5 of salt solution : NTP master mix : reagent mix : amino acids : energy mix

PLANT PATHOGEN PROTOCOLS

Fungus Genomic DNA Extraction (from roots) Protocol:

  1. Cut part of the roots (around 2 inches length) and place inside between two sink strainers
  2. Place enclosed strainers over the beaker and grind until significant root volume has been crushed and no more liquid is excreted into the beaker.
  3. Pour 40 mL of 1x TAE through the mesh sink strainers, making sure to pour over the crushed root sample.
  4. Pour resulting liquid in Falcon Tube
  5. Let the particles settle to the bottom and remove liquid.
  6. Add 400μl of distilled water to particles and shake vigorously.
  7. Pour all of the resulting mixture into a 1.5 mL microcentrifuge tube Centrifuge using OpenCellX for 10 minutes in 2 minute intervals at 5000 RPM.
  8. Incubate the solution at 65 degrees celsius with a DIY sous vide for an hour or overnight at 4°C (in the fridge).
  9. Use capillary tube to suck 1 μl of solution (first line) and release (break closed end to release) into the lid of RPA Mastermix.
  10. Use another capillary tube to suck 2.5 μl of MgOAc and release into lid of RPA Mastermix (separate from other solution)
  11. Let the DNA incubate at 37-42 degrees celsius for 20 minutes.
  12. Finally, apply the DNA to the toehold biosensor(s).
  13. A green fluorescence will indicate the presence of either Fusarium or Phytophthora, depending on which toehold was applied

REFERENCES

[1] Silverman, A. D., Kelley-Loughnane, N., Lucks, J. B., & Jewett, M. C. (2018). Deconstructing cell-free extract preparation for in vitro activation of transcriptional genetic circuitry. ACS Synthetic Biology, 8(2), 403–414. https://doi.org/10.1021/acssynbio.8b00430