Knockout1

From CSBLwiki

Contents 1 Gene replacement 1.1 Materials 1.2 Procedure 1.3 Notes

Gene replacement

• target gene: yfaL

Materials

• Plasmids
  • pKD46, pKD13, pCP20 - see this link

• Target gene
  • yfaL
    • an AmpR and CmR plasmid that shows temperature-sensitive replication and thermal induction of FLP synthesis (Datsenko and Wanner paper [1])

• Reagents
  • L-arabinose
    • Lambda red recombinase pBAD promoter inducer
• Equipment
  • Incubators (30°C and 37°C)
  • Electroporator

Procedure

• Preparation of plasmids

1. Grow up pKD46, pKD13, and pCP20 in proper host strains
2. Perform minipreps to extract plasmids

• Transformation of pKD46 to target strain
  • Preparation of competent cells
    • Using the protocol of TSS method - TSS method.
    • Target strain - BW25113, Top10

• Generation of cassette flanked by homologous arms
  • Design of primers
    • Primers have overhangs which were homologous to surrounding region of target gene.
    • Sequences from cassette region have about 60℃ Tm-value.
  • PCR condition
    • Anneling temparature and extension time depends on the target sequence.
    • Anneling temparature is tested at 55℃ at first.
    • Extension time is depend on the length of the target sequence. (1kb = about 1 min.)
  • PCR program
    • 94°C 1min → 30*[98°C 10s → 55°C 30s → 72°C 2min] → 72°C 5min

Contents	Conc.	Vol.
pKD13 template	100 ng/㎕	1㎕
forward primer	10 pmol	2㎕
reverse primer	10 pmol	2㎕
10x EX buffer		5㎕
dNTP	2 mM	2㎕
EX-tag polymerase	5unit/㎕	0.5㎕
D.W.		37.5㎕
Total		50㎕

• Treatment of PCR products

1. Add 1㎕ of DpnⅠ to not purified PCR products.
2. Incubate at 37℃ about 2 hours.
3. These samples were purified in elution buffer.

• Making competent cells

1. Pick the single colony containg pKD46.
2. Inoculate it to 5㎖ LB broth with ampicilline and L-arabinose.
   • L-arabinose final concentration is 1mM.
3. Incubate at 27℃, 1800rpm until the culture OD reaches 0.6.
4. Centrifuge 3600rpm, for 5min at 4℃.
5. Wash with ice-cold 10% glycerol 3 times.
6. Concetrate 100-fold.
   • Efficiency is

• Electroporation

1. Use 30㎕ competent cell and 300ng PCR product.
2. Pulse 2.5kV, 200Ω, 25㎌.
3. Add prewarmed 1㎖ LB broth immediately.
4. Incubate 2 hour at 37℃.
5. Spread sample and incubate at 37℃.
   • Plate on LB with 25 µg/ml Kan.
   • One-tenth portion was spread.

• Colony PCR

1. Pick the single colony
2. Resuspend in 10㎕ D.W. and
4. PCR program
   • 95°C 2min → 30*[95°C 20s → 55°C 20s → 72°C 2min] → 72°C 5min

Contents	Conc.	Vol.
template		1㎕
forward primer	10 pmol	2㎕
reverse primer	10 pmol	2㎕
10x α-tag buffer		5㎕
dNTP	2 mM	2㎕
α-tag polymerase	2.5unit/㎕	0.5㎕
D.W.		37.5㎕
Total		50㎕

• Eliminating antibiotic resistance gene
  • Transformation of pCP20 to mutant gene
    1. Add pCP20 to ice-cold 50 uL competent cell.
    2. Incubate on the ice for 20 ~ 30 min.
    3. Heat shock at 42 ºC for 1 min.
    4. After heat shock, put on ice for 2 min.
    5. Add 200 uL of pre-warmed LB broth.
    6. Shake and incubate at 30 ºC for 60 min.
    7. Plate on the pre-warmed appropriate agar plates and incubate plates at 30 ºC.
       • Plate on LB with 25 µg/ml chloramphenicol.

Notes

• Primers
  • For pKD 13 kanamycin cassette
    • Foward: ATGCGGATTATCTTTCTACGCAAGGAGTATTTATCTTTACaATTCCGGGGATCCGTCGACC
    • Reverse: gcgcccctgccagcacgtagtgtgtaggctggagctgcttcggccggttagcgccatcggct
  • For confirmation of Knockout
    • Foward: GTTTCATGCAGACTACCTGTAGTACG
    • Reverse: ctgcctcgtcctgcagttcattca

• Sequences
  • pKD13 Kanamycin cassette with FRT