Transposon-mediated Mutagenesis

Transposon-mediated Mutagenesis

Step 1 - Amplify ORF from MG1655

1.0 ul             genomic DNA (30ng/ul) 5.0 ul             gene specific primer mix 4.0 ul             2.5 mM dNTP 5.0 ul             10x Turbo Pfu buffer 0.7 ul             Turbo Pfu 34.3 ul           sterile distilled H2 O             50 ul

95°C                 95°C                55°C              72°C                72°C                   4°C 1min                 15sec               15sec               4min                  5min                 forever                                             25 cycles

Step 2 - Transposition reaction

  6 ul     ORF PCR product (~100-200ng)                                                                2 ul     Tn (Transposon, 200ng = 0.2pM)   1 ul     10x Tn reaction buffer (Epicentre)                         1 ul     Tnase (Transposase; Epicentre)                      10 ul

Vortex, spin down 37°C for 2hr Add 1 ul stop solution, 10min 70°C Purify by passing through G50 column

Step 3 � Transformation

MG1655 cells harboring the pKD46 plasmid (Datsenko and Wanner, 2000, PNAS  97(12):6640-5) are induced with arabinose to activate expression of the lRed genes and prepared for electroporation.

1. Mix following on ice:

           4.4 ul transposition reaction             40 ul MG1655 w/pKD46 electrocompetent cells

2. Transfer entire volume (~45ul) to chilled cubette (0.1cm gap), kept on ice

3. Electroporate with the following settings (for BioRad Pulse Controller)

Low Range 200 High Range 500 Capacitance (uF): -25 Total Volts: 1.8kV

4. Resuspend cells with 1ml LB and transfer contents to a 48-well growth block

5. Outgrow at 37°C for 1hr

6.  Spread entire outgrowth on a corresponding labeled LB + Amp(100ug/ml)/Kan (50ug/ml) plate in a hood and air dry.

7. Grow at 300 (to maintain pKD46). (1- 2 days)

Step 4- Picking

Day 1

1. Streak 2 colonies to single colonies on LB + Amp(100ug/ml)/Kan (50ug/ml) plates. These will be the first colonies to verify.

2. Pick 3 additional colonies into separate 96-well flat bottom plates containing 200ul Freezing media + Amp(100ug/ml)/Kan(50ug/ml) maintaining original well location.

3. Grow 300 overnight.

Day 2:

1. Grow 1 colony from each streak plate in a 96-well block with each well containing 1ml Freezing media + Amp(100mg/ml)/Kan(50mg/ml). Grow 300 overnight.

2. Freeze overnights of the other 3 plates.

Step 5� Verify mutation by Culture PCR

1. Prepare sample

Mix culture thoroughly. Transfer 20ul of culture into a 96-well plate and dilute with 80ul H2O Mix thoroughly

2. PCR Reaction.

     5 ul                         diluted culture      4 ul                         gene specific primer mix (same as in Step 1)   2.5 ul                         ExTaq premix    16 ul                        H2 O                           50 ul

95°C                 94°C            55°C                 72°C                72°C                  4°C 5min                 30sec           15 sec              4min                  5min                 forever                                           30cycles

3. Run 7 ul on 1 % test gel in 0.5x TAE to check. 

4. Analyze gel results; mutants will be ~1.2kb larger than original gene length.

Step 6 - Confirm mutations by sequencing

1. EXOSAP clean up

- Transfer 10ul of PCR product for all genes with mutant sized fragments into a separate PCR plate - Add 4ul ExoSAP-IT (USB) to each reaction - Spin briefly - React @ 37°C for 30min, then 15min@ 80°C

2. Sequence

1. Add a mix of the following to each ExoSAP reaction:

1ul of primer (KAN-2 FP-1 @ 10uM -Epicentre) 2ul Big Dye dilution Buffer (Promega) 3ul Big Dye

2. Reaction conditions: (10sec@96C ; 5sec@50C ; 4min@60C ) for 25cycles.

3. Purify through a G50 column

4. Dry plate and run on sequencer

3. Make tube stocks of confirmed mutants

Step 7 - Curing the temperature sensitive pKD46 plasmid

1. Streak confirmed mutants on a LB + Kan plate (from the above stock). Grow at 43° overnight (non-permissive temperature for pKD46 replication).

2. With a single colony inoculate the following:

1. Growth block well containing 1ml Freezing media + Kan(50ug/ml)/well . 2. LB+Kan agar in a 96 well plate 3. LB+Amp agar in a second 96-well plate

3. Grow overnight at 37°C. Cured cells will grow on Kan but not on Amp.

4. Confirm by PCR and sequencing as above.