TITLE: DatsenkoWannerCportucalensis
AUTHORS: Dianne K Newman, Lev Tsypin, Scott Saunders, Allen W Chen

[DESCRIPTION]
Protocol for performing Datsenko-Wanner deletions of C. portucalensis MBL genes based on https://doi.org/10.1073/pnas.120163297

[STEPS]
SECTION: Amplify the resistance cassette for knocking out a genetic region of interest using the appropriate primer pairs. Use a high-fidelity polymerase, such as Q5, KAPA, or Phusion according to manufacturer instructions.
1. Design primers for replacing the gene locus of interest with a Kanamycin resistance casette via homologous recombination.
SECTION: Amplify the resistance cassette for knocking out a genetic region of interest using the appropriate primer pairs. Use a high-fidelity polymerase, such as Q5, KAPA, or Phusion according to manufacturer instructions.
1.1. Design primers with 36 bp homology upstream to/downstream from region you want to knock out.
SECTION: Amplify the resistance cassette for knocking out a genetic region of interest using the appropriate primer pairs. Use a high-fidelity polymerase, such as Q5, KAPA, or Phusion according to manufacturer instructions.
1.2. Append GTGTAGGCTGGAGCTGCTTC to 3' end of upstream primer.
SECTION: Amplify the resistance cassette for knocking out a genetic region of interest using the appropriate primer pairs. Use a high-fidelity polymerase, such as Q5, KAPA, or Phusion according to manufacturer instructions.
1.3. Append CATATGAATATCCTCCTTAG to 3' end of downstream primer.
SECTION: Amplify the resistance cassette for knocking out a genetic region of interest using the appropriate primer pairs. Use a high-fidelity polymerase, such as Q5, KAPA, or Phusion according to manufacturer instructions.
1.4. Use pKD4 for KanR cassette template.
SECTION: Amplify the resistance cassette for knocking out a genetic region of interest using the appropriate primer pairs. Use a high-fidelity polymerase, such as Q5, KAPA, or Phusion according to manufacturer instructions.
2. Use Phusion polymerase for PCR: approx. 15 ng plasmid template per reaction

1) 98 ºC 30 sec.
2) 98 ºC 5 sec.
3) 55 ºC 30 sec.
4) 72 ºC 54 sec.
5) Go to step 2 24 times
6) 72 ºC 5 min.
7) hold at 22 ºC or refrigerate/freeze until use


SECTION: Amplify the resistance cassette for knocking out a genetic region of interest using the appropriate primer pairs. Use a high-fidelity polymerase, such as Q5, KAPA, or Phusion according to manufacturer instructions.
3. Purify PCR products using NEB PCR cleanup kit (Cat. No. T1030) after gel verification.
SECTION: Deletion of genomic region
5. Plate 300 µL of recovered cell suspension on LB + 50 µg/mL kanamycin and grow at 37 ºC. Leave remaining cell suspension standing overnight and plate the next day if nothing grows from the first attempt.
SECTION: Deletion of genomic region
6. Cure pKD46 plasmid and confirm deletion
SECTION: Deletion of genomic region
6.1. Pick a couple individual colonies for each transformation and re-streak on non-selective LB to grow at 42 ºC overnight (pKD46 has a temperature-sensitive origin of replication).
SECTION: Deletion of genomic region
6.2. From each re-streak, pick a single colony to patch onto non-selective LB, LB/Kan, and LB/Carb or Amp. Grow at 30 ºC.
SECTION: Deletion of genomic region
6.3. Check for colonies that are kanamycin-resistant and carbenicillin/ampicillin-sensitive.
SECTION: Deletion of genomic region
6.4. PCR verify correct insertion of kanamycin resistance cassette (forward primer in KanR and reverse downstream from knocked out region and loss of λRed (primers in λRed gene on pKD46).
SECTION: Deletion of genomic region
6.5. Prepare -80C stocks for good strains in 35% glycerol from cured overnight cultures grown in LB + 50 µg/mL kanamycin.
SECTION: Deletion of genomic region
7. FLP out resistance cassette
SECTION: Deletion of genomic region
7.1. Electroporate pCP20 (AmpR, ts-origin) into cured deletion strains. See: dx.doi.org/10.17504/protocols.io.kqdg3x7r7g25/v1
SECTION: Deletion of genomic region
7.2. Recover in LB at 30 ºC for 1 hour.
SECTION: Deletion of genomic region
7.3. Plate 100 µL to grow overnight on LB/Carb or Amp plate at 30 ºC.
SECTION: Deletion of genomic region
7.4. Streak several colonies on non-selective LB and grow at 42 ºC overnight (the pCP20 plasmid also has a temperature-sensitive origin of replication).
SECTION: Deletion of genomic region
7.5. Screen for loss of all antibiotic resistance by patching on non-selective and selective LB plates.
SECTION: Deletion of genomic region
7.6. Confirm FRT scar by PCR using primers in flanking regions (e.g., ∆nap_check_F and ∆nap_check_R) and sequencing.
SECTION: Deletion of genomic region
4.11. Transfer cell suspension to microcentrifuge tube and recover at 37 ºC shaking horizontally for an hour.
SECTION: Deletion of genomic region
4.10. Add 500 µL LB to each cuvette.
SECTION: Deletion of genomic region
4.9. Electroporate in ice-cold cuvettes using 2.5 kV, 250 Ω, and 25 µF (confirm approx. 5 ms pulse for each), assuming 2 mm gap cuvettes.
SECTION: Deletion of genomic region
4.8. Add approximately 100 ng resistance cassette to cell aliquot and gently mix by tapping.
SECTION: Deletion of genomic region
4.7. Aliquot 100 µL reaction volumes into ice-cold microcentrifuge tubes.
SECTION: Deletion of genomic region
4.6. Resuspend into 400 µL final vol 10% glycerol (assuming four reactions--100 µL per reaction).
SECTION: Deletion of genomic region
4.5. Wash at 2000 x g (slow deceleration) at 4 ºC into ice-cold 10% glycerol three times, combining final aliquot in one tube during last wash.
SECTION: Deletion of genomic region
4.4. Grow at 30 ºC to OD600 approx. 0.6-0.8 (around 2 hours).
SECTION: Deletion of genomic region
4.3. Morning of, inoculate 2 mL overnight culture into 100 mL (25 mL per transformation) LB with 50 µg/mL Amp/Carb and 0.2% L-arabinose.
SECTION: Deletion of genomic region
4.2. One day prior, grow overnight culture in 5 mL LB with 50 µg/mL Amp/Carb.
SECTION: Deletion of genomic region
4.1. Two days prior, streak out C. portucalensis MBL/pKD46 on 50 µg/mL ampicillin (Amp) or carbenicillin (Carb) LB agar at 30 ºC.
SECTION: Deletion of genomic region
4. Electroporate the antibiotic resistance casette (PCR product from Step 3) into C. portucalensis MBL carrying pKD46 (the λRed plasmid)