Protocol for gene knockout

Day 1

A. Transform pKD46 into (chemically competent) target strain

  1. Trasform pKD46 into the host strain, following the instructions from the wiki for Bacterial Transformation of Chemically Competent Cells
  2. Grow cells on LB+Amp plates o/n at 30°C
  3. Make glycerol stock.

B. PCR amplify linear fragment from pKD3 or pKD4

Note: these PCR reaction volumes are for Pfx Platinum DNApolymerase. You will want to adjust your PCR if using another DNA Pol

  1. See Primer Design page here.
    • Takes about 3days to order 70nt oligos from IDTdna.org
    • Make sure to dissolve each primer stock to 100μM in ddH2O.
    • Add oligo to OligoRegistry worksheet.
  2. Make master mix:…………..μl/rxn
    1. 10x PCR buffer……..25
    2. 10mM dNTPs…………3
    3. 50mM Mg2SO4………2
    4. 10x enhancer……….30
    5. ddH2O………………..34
    • Note: mix together {n+1} volumes of each substrate, where n=the number of reactions you will be doing.
    • Note: these volumes are for 100uL reactions. Adjust if using lower volumes.
  3. Make 10μM primer mix:
    1. Mix 2μL of both primers (100μM stock) into 18μL ddH2O.
    2. If you need more than 20μL of primer, adjust volumes. (1μL of each per total 10μL mix)
  4. For each reaction, mix together in PCR tube.
    1. 94μL Master Mix
    2. 1μL Template DNA (either pKD3 for ChlR or pKD4 for KanR)
    3. 3μL Primer mix (10μM each primer)
    4. 2μL Pfx Platinum DNA Pol
  5. Program cycle in PCR thermocycler with steps 2-4 repeating 34 times.
    1. 94°C at 5:00 (m:s)
    2. 94°C at 0:15
    3. 55°C at 0:30 (even if the Ta is higher, use 55°C)
    4. 68°C at 2:00
    5. 68°C at 7:00
    6. 4°C at ∞
  6. PCR purify.
  7. Digest template with DpnI
    • Note: DpnI only digests methylated DNA. This digestion will knockout any residual plasmid so that it will not interfere with the electroporation of the PCR fragment.
    1. Volumes as follows:
      • 28μL PCR purified sample (the entire sample)
      • 3.5μL NEB 10x buffer #4
      • 0.3μL DpnI enzyme
      • 5.2μL H2O
    2. Incubate at 37°C for 1 hour.
  8. PCR purify again. Check products on a gel.
    • If using pKD3, the product should be 1.1kb
    • If using pKD4, the product should be 1.6kb

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Day 2

Make {target strain, pKD46} electrocompetent

There are a couple of tricks to making cells electrocompetent. (1) Remove as much salt from the cell suspensions as possible to prevent arcing. (2) The cells must remain cold (either 4°C cold room or on ice). Once in DI water, the cells become very sensitive to temperature changes and the transformation efficiency drops dramatically if cells are allowed to warm up above 4°C at any step.

In this case, we have to make the cells “fresh” because they are induced. If you’d like to make a large-scale electrocompetent cells, please see xxx.

  1. Pre-chill all tubes, solutions, and cuvettes!
  2. Pick some colonies from a fresh plate (or back dilute with 20uL from o/n culture) and grow at 30degC in 2mL LB+Amp.
    • Note: Include enough samples for +/- L-arabinose induction
  3. When OD600 = 0.1, add 20uL of L-arabinose stock to induce pKD46 λ-red expression
  4. Continue growing at 30degC until OD600 = 0.4 – 0.6
  5. Aliquot 1mL from each sample into 2x 1.5mL centrifuge tubes
  6. Chill cells in ice-water bath 10-15min
  7. Centrifuge 10m at 4000rcf at 4°C
    • Note: the centrifuge next to the bioflo cabinet has temp control
  8. Pipette/discard supernatant and resuspend cells (GENTLY) in 1mL ice-cold dH2O
  9. Centrifuge 10m at 4000rcf at 4°C
  10. Resuspend cells (GENTLY) in 0.5mL ice-cold dH2O
    • Note: after washing in DI water, the cells become more and more difficult to spin down. Using 10% glycerol can help spin down cells
  11. Centrifuge 10m at 4000rcf at 4°C
  12. Resuspend pellet (GENTLY) in 50uL ice-cold water.

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Electroporate linear PCR product into electrocompetent cells

  1. Chill cuvettes for at least 5 minutes on ice.
  2. Set electroporator to 2.5kV. (Our electroporator is set to 600Ω)
  3. Add 0.5ug DNA to cells. Mix by pipetting. Pippet into a sterile e.p. cuvette.
    • Note: This DNA should be salt-free, so when purifying with kit, use water.
    • Note: Include enough samples for +/- PCR.
    • Note: The DNA should not sit in the cells for more than 1 minute.
  4. Make sure to dry the cuvette to prevent arcing!
  5. Place the DRY cuvette into the sample chamber. Apply the pulse by pushing the pulse button twice. Remove the cuvette.
    • Note: If you hear a popping sound, this means that the sample has arced. This could either be (1) too much salt in your sample or (2) a wet cuvette.
  6. Immediately add 1mL of room-temp SOC or LB and transfer to a culture tube
  7. Incubate 1 hr with low shaking at 37°C. (Not 30°C…you don’t need to keep pKD46 around anymore)
  8. Plate out cells on LB+antibiotic. (Chl for pKD3, Kan for pKD4) Grow 24hr at 37°C.

HUGE HINTS, MY FRIEND!

  • When you plate out the cells, use super-dooper-low antibiotics (5ug/mL). You’ve just shot your cells with a high voltage, and now their only way to survive the sting of antibiotics is to do this complicated recombination with foreign proteins…give your cells a break…low antibiotics are the trick!
  • See “Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes” by Maite Muniesa in BMC Molecular Biology for more hints.
  • Keep your electroporation cells o/n (either in more LB at 37°C or in the 4°C fridge) just in case they take a bit longer to survive.

hi.

   I am xingling jiang from Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences.
   I am doing the red knockout in BW25113 following your protocol released on openwetware. u said "When you plate out the cells, use super-dooper-low antibiotics (5ug/mL)." I used 10ug/ml Cm(i think it is already very low), but that resulted in about one hundred of clonies for negative control(- PCR, 500ul was plated). +pcr samples had more clonies(about 150-200,500ul plated),however none of them passed the PCR verification. like this i have failed several times.
    Here is my questions.
      Is it right to get so many clonies on -pcr negative control plate?
      How many clonies do u get usually?How much is the positive rate? e.g. How many clonies can pass the deletion pcr verification?
      I am the fist one in my lab. doing the red knockout. I know something must be wrong but just don't know what it is. Thank u for your help in advance.

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Day 3

Verify deletion with colony PCR

Do colony PCR with select primers to verify that the Antibiotic resistance and FLP sites are present.

  1. Pick colonies
    1. Pick colonies with a pipette tip and resuspend in 20 μl of cold ddH2O by pipetting up and down
    2. Pipette 3μl onto an index plate with appropriate antibiotic for use later if colony is good. Make sure to not plunge down near the plate because this will cause “splattering” of cells.
    3. Grow index plate at 37°C o/n.
  2. Make master mix:…………..20 μl/rxn
    1. 10x PCR buffer……….5.0
    2. 10mM dNTPs…………0.6
    3. 50mM Mg2SO4……….0.4
    4. 10x enhancer…………6.0
    5. ddH2O…………………3.0
    • Note: mix together {n+1} volumes of each substrate, where n=the number of reactions you will be doing.
    • Note: these volumes are for 20uL reactions. Adjust if using lower volumes.
  3. Make 10μM primer mix:
    1. This time, use the shorter(25bp) primers.
    2. Mix 2μL of both primers (100μM stock) into 16μL ddH2O.
    3. If you need more than 20μL of primer, adjust volumes. (1μL of each per total 10μL mix)
  4. For each 20 μl reaction, mix together in PCR tube.
    1. 15μL Master Mix
    2. 2.0μL Colony suspension (template)
    3. 2.0μL Primer mix (10μM each primer)
    4. 1.0μL Pfx Platinum DNA Pol
  5. Program cycle in PCR thermocycler with steps 2-4 repeating 34 times.
    1. 94°C at 5:00 (m:s)
    2. 94°C at 0:15
    3. 55°C at 0:30
    4. 68°C at 2:00
    5. 68°C at 7:00
    6. 4°C at ∞
  6. Check products on a gel with 10μL samples (2.5μL 5xdye). Should be the same size as the PCR product from earlier. Also–run a control using the host strain with pKD46. This should result in the length of the gene(s) to be knocked out+100.

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Day 4

Remove antibiotic resistance with pCP20

To get rid of the antibiotic resistance, electroporate with pCP20.

  1. Pre-chill all tubes, solutions, and cuvettes!
  2. Grow host strain with knocked out gene-of-interest and added antibiotic resistance gene in antibiotics at 37°C until OD600 = 0.4 – 0.6.
  3. Aliquot 1mL from each sample into 2x 1.5mL centrifuge tubes

Chill cells in ice-water bath 10-15min

  1. Centrifuge 10m at 4000rcf at 4°C
    • Note: the centrifuge next to the bioflo cabinet has temp control
  2. Pipette/discard supernatant and resuspend cells (GENTLY) in 1mL ice-cold dH2O
  3. Centrifuge 10m at 4000rcf at 4°C
  4. Resuspend cells (GENTLY) in 0.5mL ice-cold dH2O
    • Note: after washing in DI water, the cells become more and more difficult to spin down. Using 10% glycerol can help spin down cells
  5. Centrifuge 10m at 4000rcf at 4°C
  6. Resuspend pellet (GENTLY) in 50uL ice-cold water.
  7. If pCP20 was mini-prepped in a buffer, dilute it down in pure water to minimize salt effects while electroporating.
  8. Electroporate cells with x-0.5pg DNA. Set electroporator to 2.5kV. (Our electroporator is set to 600Ω)
  9. Immediately add 1mL SOC or LB to the cuvette, resuspend cells, transfer to a culture tube.
  10. Grow at 30°C for 1-3 hours.
  11. Plate 200μL of cells on Amp plates and 200μL of cells on Kan plates. Grow at 30°C for 16-24 hours.
    • The Amp plate will have the cells with pCP20.
    • If low [Amp] plates aren’t available, or if you don’t see colonies after an overnight growth, continue growing for up to another 24 hours. Amp is susceptible to satellite colonies, so it’s more likely than larger colonies have pCP20 rather than smaller ones.
    • The Kan plate will give an idea of if the cells survived the electroporation
  12. Colony purify a few transformants non-selectively (no antibiotics) at 43°C.
    • USE GOOD STERILE TECHNIQUE
    • Make an index plate on Amp at 30°C. If no colony shows on this plate, scrap that colony from the 43°C plate.

Day 5

Test the colony purified transformants for sensitivity to Amp and Kan.

  1. Pick a colony from the 43°C plate with a pipette tip and resuspend in 10μL LB.
  2. Pipette 3μL of the cell suspension onto a set of 3 plates:
    1. Kan, 37°C
    • Kan will test for the presence/absence of KanR gene in the genome of the host strain.
    1. Amp, 30°C
    • Amp will test for the presence/absence of AmpR from the pCP20 plasmid.
    1. LB only, 37°C
    • If there is no growth in neither the Kan or Amp plate for a particular colony, it has lost both the KanR gene in the genome as well as the AmpR from pCP20.
  3. Pick a “good” colony and re-streak it on an LB plate to get single colonies. Grow o/n at 37°C.

Day 6

Colony PCR for scar

To confirm that the KanR gene has been removed by pCP20, do a colony PCR on the hostΔgene colony. This is the same exact procedure as above. When doing the colony PCR make sure to

  1. make an index plate (0 antiobiotics, 37°C)
  2. start an o/n culture at 37°C. This culture can subsequently be used to make a glycerol stock AND a large batch of chemically competent cells.

See the procedure for large-batch electrocompetent cell prep here.

Glycerol stocks to keep

Note that it may also be in your best interest to make several either chemically competent or electrocompetent frozen stocks for future work. ALSO — make sure to make glycerol stocks of ALL STRAINS in case contamination happens along the way.

  1. Host+pKD46
  2. HostΔGene+Kan/Chl
  3. HostΔGene+Kan/Chl + pCP20
  4. HostΔGene (final product)

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