Recombineering/Lambda red-mediated gene replacement

Overview

Single-gene knockouts using λ red system, adapted from Datsenko and Wanner paper. The goal of this protocol was to create an endA (coding for endonuclease I) knockout, but obviously it can be adapted to any gene. The knocked-out gene is replaced with an antibiotic resistance gene, usually for kanamycin or chloramphenicol. In this example, the target strain was already kanamycin resistant, so the chloramphenicol resistance gene was used.

Materials

• plasmids
  • pKD46
  • pKD3 (chloramphenicol)
  • pKD4 (kanamycin)
  • pCP20 (optional)
• reagents
  • L-arabinose
• equipment
  • incubators (30[[:Category:{{{1}}}|{{{1}}}]] and 37[[:Category:{{{1}}}|{{{1}}}]])
  • electroporator

Procedure

• Geneeral outline
  • Grow up pKD46, pKD3, and pCP20 in current host strains
  • Perform minipreps to extract plasmids
  • Transform pKD46 into target strain
  • PCR amplify linear fragment from pKD3 using oligos A and B (see below for design)
  • Make target strain (now maintaining pKD46) electrocompetent by growing at 30[[:Category:{{{1}}}|{{{1}}}]] with L-arabinose
  • Electroprate linear DNA into electrocompetent cells
  • Grow at 37[[:Category:{{{1}}}|{{{1}}}]] on chloramphenicol plates
  • PCR verify the deletion with oligos C and D (see below for design)
• Detailed procedure
  • This was my first attempt at this protocol, and I left out many proper controls. Consider planning a little better.
• Day 0: Start overnight culture
  • Start overnight culture of strain containing gene to knock out.
• Day 1: Preparation and transformation of competent cells
  • Make new glycerol stock of overnight strain (grown from single colony)
  • Add 300 μL overnight culture to 30 mL LB medium (1:100 dilution)
  • Check density every 30 minutes starting at +1 hour; grow to OD₆₀₀ of 0.3 to 0.4
  • OD₆₀₀ measurements
    • +2:00 hrs: 0.06
    • +2:45 hrs: 0.3008
  • Spin at 2500rcf for 10 minutes at 4[[:Category:{{{1}}}|{{{1}}}]] in two 50 mL centrifuge tubes (JA-20 rotor)
  • Decant supernatant, discard
  • Resuspend each pellet in 5 mL ice cold transformation buffer; swirl or pipette gently to mix
    • Transformation Buffer
    • 10 mM Pipes
    • 15 mM CaCl₂
    • 250 mM KCl
    • Titrate to pH 6.7 before adding MnCl₂
    • 55 mM MnCl₂
    • Filter sterilize
  • Incubate on ice for 10 min
  • Spin at 2500rcf for 10 minutes at 4[[:Category:{{{1}}}|{{{1}}}]]
  • Decant supernatant, discard
  • Resuspend each pellet in 1.25 mL ice cold transformation buffer
  • Combine resuspended pellets in single tube
  • Remove 400 μL for immediate transformation
  • Add DMSO to a final concentration of 7% (160 μL). Drip the DMSO slowly into the cell suspension, with constant swirling by hand.
  • Incubate on ice for 10 min
  • Aliquot 400 μL each into five 1.5 mL tubes
  • Store in -80[[:Category:{{{1}}}|{{{1}}}]] freezer.
  • Transform strain with pKD46 and grow on LB-amp-kan plate at 30[[:Category:{{{1}}}|{{{1}}}]]
    • Prepare four tubes with 0, 1 ng, 10 ng, and 100 ng pKD46 plasmid DNA
    • Add 100 μL of competent cell mix to each tube
    • Incubate on ice 30 min
    • Heat shock 30 seconds at 42[[:Category:{{{1}}}|{{{1}}}]]
    • Incubate on ice 2 min
    • Spread all 100 μL on LB-amp-kan plate
    • Incubate at 30[[:Category:{{{1}}}|{{{1}}}]] overnight
  • PCR endA-CmR from pKD3

Contents	Concentration	Volume
pKD3 template	45 ng/μL	0.5 μL
forward primer	10 μM	5 μL
reverse primer	10 μM	5 μL
10x KOD buffer	-	10 μL
dNTP	2 mM	10 μL
MgSO4	25 mM	4 μL
KOD polymerase		2 μL
dH2O	-	64.5 μL

• • PCR program: 95[[:Category:{{{1}}}|{{{1}}}]] 7min → 20*[94[[:Category:{{{1}}}|{{{1}}}]] 15s → 50[[:Category:{{{1}}}|{{{1}}}]] 30s → 72[[:Category:{{{1}}}|{{{1}}}]] 90s]

Notes

Designing necessary primers

• First look at the sequence of the plasmid containing the resistance marker you wish to swap in for your target gene.
  • NCBI sequence viewer: pKD3
    • priming site 1: GTGTAGGCTGGAGCTGCTTC
    • priming site 2: GGACCATGGCTAATTCCCAT
    • priming site 2 reverse complement: ATGGGAATTAGCCATGGTCC

• pKD3 CmR sequence (1033 bases)

GTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCTTTAAATGGCGCGCCTTACGCCCCGCCC TGCCACTCATCGCAGTACTGTTGTATTCATTAAGCATCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTC GCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGA GACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTC GTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGC CATACGTAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAAT ATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGT GATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGACAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCT TACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCAC AGGTAGGCGCGCCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTAAGGAGGATATTCATATGGACCATGGCTAATTCCCAT

• Next, find the sequence (and context sequence) of the gene you wish to remove
  • In this example, I found the sequence and context for endA (808 bases total)
    • MG1655_m56_ABE-0009661 +50bp upstream +50bp downstream

CCAAAACAGCTTTCGCTACGTTGCTGGCTCGTTTTAACACGGAGTAAGTGATGTACCGTTATTTGTCTATTGCTGCGGTGGTACTGAGCGCAGCATTTTCCGGC CCGGCGTTGGCCGAAGGTATCAATAGTTTTTCTCAGGCGAAAGCCGCGGCGGTAAAAGTCCACGCTGACGCGCCCGGTACGTTTTATTGCGGATGTAAAATTAA CTGGCAGGGCAAAAAAGGCGTTGTTGATCTGCAATCGTGCGGCTATCAGGTGCGCAAAAATGAAAACCGCGCCAGCCGCGTAGAGTGGGAACATGTCGTTCCCG CCTGGCAGTTCGGTCACCAGCGCCAGTGCTGGCAGGACGGTGGACGTAAAAACTGCGCTAAAGATCCGGTCTATCGCAAGATGGAAAGCGATATGCATAACCTG CAGCCGTCAGTCGGTGAGGTGAATGGCGATCGCGGCAACTTTATGTACAGCCAGTGGAATGGCGGTGAAGGCCAGTACGGTCAATGCGCCATGAAGGTCGATTT CAAAGAAAAAGCTGCCGAACCACCAGCGCGTGCACGCGGTGCCATTGCGCGCACCTACTTCTATATGCGCGACCAATACAACCTGACACTCTCTCGCCAGCAAA CGCAGCTGTTCAACGCATGGAACAAGATGTATCCGGTTACCGACTGGGAGTGCGAGCGCGATGAACGCATCGCGAAGGTGCAGGGCAATCATAACCCGTATGTG CAACGCGCTTGCCAGGCGCGAAAGAGCTAACCTACACTAGCGGGATTCTTTTTGTTAACCCCTACCCCACGCGTACAACC

• Construct primers that have internal overlap with the resistance marker (pKD3) and external overlap with the target knockout gene (endA).
  • Forward primer: A
    • CCAAAACAGCTTTCGCTACGTTGCTGGCTCGTTTTAACACGGAGTAAGTGGTGTAGGCTGGAGCTGCTTC
  • Reverse primer: B
    • GGTTGTACGCGTGGGGTAGGGGTTAACAAAAAGAATCCCGCTAGTGTAGGATGGGAATTAGCCATGGTCC

• Construct primers that only flank the target gene (endA) for PCR verification
  • Forward primer: C
    • CCAAAACAGCTTTCGCTACGTTGCT (25 bases)
  • Reverse primer: D
    • GGTTGTACGCGTGGGGTAGGGGTTA (25 bases)

• Figure out the sequence and size of what you should expect if everything works. In this case, it's CmR inserted into endA flanking region (1132 bases total)

CCAAAACAGCTTTCGCTACGTTGCTGGCTCGTTTTAACACGGAGTAAGTGGTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTC GGAATAGGAACTTCATTTAAATGGCGCGCCTTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTATTCATTAAGCATCTGCCGCATGGAAGCCATCACAAA CGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGC CACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAAC ACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAA CAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGTAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGG ATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAA ATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGACAACTCAAAAAA TACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTAT CAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTAGGCGCGCCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGG AACTAAGGAGGATATTCATATGGACCATGGCTAATTCCCATCCTACACTAGCGGGATTCTTTTTGTTAACCCCTACCCCACGCGTACAACC

References

λ red Links

• Schematic Overview
• Protocol from Charlie Kim

endA Links

• Entrez Gene: endA
• ASAP annotation for K12 endA

1. Datsenko KA and Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5. DOI:10.1073/pnas.120163297 |

Contact

• Shawn Douglas — posted original protocol, performed once in August 2006.