Goal

Vector map of the pKD46 plasmid. pKD20 was used in the original publication, but pKD46, constructed by the same authors, shows higher recombination efficiency and is the one used in all recombination experiments.

Vector map of the pET-21c plasmid.

MCS of pET-21c with the binding sites of the IK07, IK08, and IK02 primers highlighted.

Integration of the methylmalonyl-CoA sythesis pathway into the BAP1 genome. This is a reproduction of a part of <bib id="pmid17959404"/>. For the integration, the λ-red protocol established by Datsenko and Wanner<bib id="pmid10829079"/> is used. pKD20/pKD46 plasmids contain the λ-red genes begind an arabinose inducible promoter, a temperature-sensitive origin, and an ampicillin selection marker. The part to be integrated is on the pLF03 plasmid, which is identical to pYW201<bib id="pmid17959404"/> in every way except for the selection marker in the insert (pYW201 has kanamycin resistance, pLF03 chloramphenicol). pLF03 contains an ampicillin marker that is expressed constitutively, chloramphenicol is only used to test for successful integration, as it is behind an IPTG-inducible T7 promoter (as are the other two genes, pccB and accA1). pLF03 is derived from pET-21c. The expected amplificate size for PCR of pLF03 is 4kb. pccB-accA will replace the ygfG gene in the BAP1 genome.

BL21-DE3 genomic region around ygfG, which will be replaced by pccB-accA in BAP1. Sites homologous to ygfG21C1 and ygfG21C2 as well as the IK01 primer binding site are shown.

2013-06-10

Gel electrophoresis of the pLF03 digestion. Left: NEB 2 log DNA ladder; middle: digestion with EcoRI, right: digestion with XhoI+NdeI

• need to verify plasmid identity: digest pLF03 with EcoRI (for linearization) and XhoI+NdeI (verify insert size); 388 ng (4 µl of miniPrep from 2013-06-04) each
• insert only 3.3 kb in length, expected >4 kb ([http://www.uniprot.org/uniprot/Q9RGQ6 AccA1]: 590AS * 3 = 1770 bp, [http://www.uniprot.org/uniprot/Q9X4K7 PccB]: 530AS * 3 = 1590 bp, [http://www.uniprot.org/uniprot/P62577 CatR]: 219 AS = 657 bp; adds up to 4017 bp, not counting RBS and spacers between the individual genes)
• contacted Lei Fang (Pfeifer Lab), there are NdeI and EcoRI sites in catR (they were not indicated in his plasmid map)
• to verify presence of chloramphenicol acetyl transferase in insert: transform BAP1 with 97 ng pLF03 (1 µl of miniPrep from 2013-06-04), plate on Amp + IPTG
• evening: pick BAP1-pLF03 colonies, transfer to Cm + IPTG
• inoculate 4 and 5 ml of liquid culture (Amp) with BAP1-pLF03

2013-06-11

Gel electrophoresis of the PCR amplificate. Lane 1: NEB 2-log ladder; lane 2: PCR with Phusion Flash; lane 3: PCR with Taq at 43°C annealing temperature; lane 4: PCR with Taq at 41.5°C annealing temperature

• BAP1-pLF03 grew on Cm + IPTG
• try to optimize PCR:
• run PCR of 5 ng pLF03 (1 µl 1:20 dilution of miniPrep from 2013-06-04) with primers ygfG21C1 and ygfG21C2 using Phusion Flash F548 polymerase (performed by Dominik):

• run PCR of 5 ng pLF03 (1 µl 1:20 dilution of miniPrep from 2013-06-04) with primers ygfG21C1 and ygfG21C2 using Taq polymerase (cheaper):

• prepare glycerol stock of BAP1-pLF03
• make miniPreps of BAP1-pLF03 -> 21 ng / µl and 27 ng / µl in 27.5 µl

• received just plated DH5α-pCP20 from Sourjik Lab, grow at 30°C

2013-06-12

• purify Phusion Flash PCR product with Qiagen PCR purification kit -> 120 ng / µl in 27.5 µl
• inoculate 2 x 5 ml LB + Amp with DH5α-pCP20, grow at 30°C

2013-06-13

• miniPreps of DH5α-pCP20 -> 7.8 ng / µl and 14.6 ng / µl in 27.5 µl, respectively
• low yield of miniPreps: inoculate 2 x 5 ml LB + Amp with DH5α-pCP20, grow at 30°C

2013-06-14

• miniPreps of DH5α-pCP20 -> 25 ng / µl and 29 ng / µl in 27.5 µl, respectively
• prepare glycerol stocks of DH5α-pCP20
• to avoid electroporation with intact pLF03: add 10 µl of PCR amplificate (1.2 mg) on gel, perform gel extraction (QiaGen gel extraction kit) -> 3.2 ng / µl
• add 240 ng (2 µl) raw PCR product to 100 µl electrocompetent BAP1-pKD46
• add 16 ng (5 µl) gel-extracted PCR product to 100 µl electrocompetent BAP1-pKD46
• electroporate 50 µl each, add to 1 ml SOC with 1 mM IPTG
• shake at 300 rpm, 37°C for 1 h
• spin cells down (3 min 10 000 rpm = 8000 g), decant supernatant (leave about 200 µl supernatant in eppendorf tube)
• resuspend pellet, plate 100 µl on Cm-IPTG, leave rest at RT
• grow at 37°C

2013-06-15

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG. Lane 1: NEB 2-log; lane 2: negative control (BAP1-pLF03); rest: colony-PCRs from PCR-purified plate.

• one colony on plate with gel-extracted PCR product
• many colonies on plate with PCR-purified PCR product
• pick colonies from plate with PCR-purified PCR product, run colony-PCR (Taq, 25 µl total volume) with primers IK01 and IK02 (expected product: 465 bp):

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG. Lane 1: NEB 2-log; lane 2: negative control (BAP1-pLF03); lane 3: colony-PCR of colony electroporated with gel-extracted PCR product; rest: colony-PCRs of colonies electroporated with PCR-purified PCR product.

• no product -> pick colony from gel-extracted plate, pick colonies from PCR-purified plate, run colony-PCR (Taq, 25 µl total volume) with primers IK01 and IK02:

• no product -> 2 possibilities:
  1. all colonies bear complete pLF03 plasmid (PCR was run with 5 ng template, DNA concentration in PCR product: 120 ng / µl, electroporated with 2 µl -> 1/6 ng pL03 vs. 240 ng amplificate)
  2. PCR did not work
• for colony from gel-extracted plate + 3 colonies from PCR-purified plate: add up to 3 ml medium, add IPTG (1 mM) + Cm, grow at 37°C
• plate rest of bacteria electroporated with gel-extracted PCR product on Cm + IPTG

2013-06-16

miniPrep of BAP1-pKD46 electroporated with PCR product from pLF03. Lane 1: NEB 2-log; lane 2: miniPrep of colony electroporated with gel-extracted product; rest: miniPreps of colonies electroporated with PCR-purified product.

• no colonies on gel-extracted plate
• make miniPreps of ON cultures, run gel
• pLF03: 9.2 kb, bands above 10 kb, bands smear, low intensity (applied all 30 µl of miniPrep to gel) -> fragments of genomic DNA?
• => PCR went wrong?

2013-06-17

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG. Lanes 1-6: annealing at 66°C; lanes 8-11: touch-down 66->60°C; lane 6: NEB 2-log; lane 1,7: negative control (BAP1-pLF03); lane 2,8: colony-PCR of colony electroporated with gel-extracted PCR product; rest: colony-PCRs of colonies electroporated with PCR-purified PCR product.

• repeat colony PCR, use 1 µl of liquid culture from gel-extracted plate, pick colonies from PCR-purified plate (Taq, 25 µl total volume)
• 2 conditions:

• no positives
• plate samples of liquid cultures from 2013-06-15 on Amp, grow at 37°C

2013-06-18

• colonies grew on Amp

2013-06-19

Gel electrophoresis of the PCR amplificate. Lane 1: NEB 2-log ladder; lanes 2-5: PCR with Phusion Flash (2 µl of product were put on gel)

• prepare for repetition of experiment: run 4 PCRs of 1 ng pLF03 (miniPrep from 2013-06-04) with primers ygfG21C1 and ygfG21C2 using Phusion Flash (performed by Dominik), one reaction = 50 µl total volume

Gel electrophoresis of the purified PCR amplificate. Lane 1: NEB 2-log ladder; lane 2: purified PCR with Phusion Flash (1 µl of product were put on gel)

• pool products, purify (digestion only 3 h)
• nanoDrop: 2088 ng/µl -> remeasure: 532 ng/µl
• load 1 µl purified PCR product on gel -> very weak band -> nanoDrop wrong
• to eliminate error: make fresh electrocompetent cells:
  • pick colony from BAP1-pKD46 plate from 2013-06-04
  • inoculate 1 ml LB + Amp
  • grow at 30°C

2013-06-20

• too little cells: use aliquot from 2013-06-06
• use all available DNA (14 µl) and 100 µl cells (complete aliquot)
• grow in SOC + IPTG(1mM) at 300 rpm for 3h
• spin cells down, decant supernatant, resuspend in remainder of medium
• plate 10 µl, rest on Cm + IPTG

2013-06-24

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG. Lane 1: NEB 2-log; lanes 2-4: negative control (BAP1-pLF03); lanes 5-10: colony-PCR of colony electroporated with purified PCR product; lanes 2,5,8: primers IK01+IK02; lanes 3,6,9: primers IK01+IK03 (positive control); lanes 4,7,10: primers IK05+IK06 (negative control)

• no colonies on 10 µl plate, 2 colonies on rest plate
• colony-PCR using primer pairs IK01+IK02, IK01+IK03 (positive control), IK05+IK06 (negative control) (Taq, 20 µl total volume)

• inconclusive results: no band for IK05+IK06 in BAP1-pLF03 (negative control)
• grow liquid cultures at 37°C

2013-06-25

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG and then in LB ON. Lane 1: NEB 2-log; lanes 2-5: negative control (BAP1-pLF03); lanes 6-13: colony-PCR of colonies electroporated with purified PCR product; lanes 2,6,10: primers IK01+IK02; lanes 3,7,11: primers IK01+IK03 (positive control); lanes 4,8,12: primers IK05+IK06 (negative control); lanes 5,9,13: primers IK01+IK06 (negative control)

• repeat PCR with 1 µl of ON cultures:

• genomic integration failed
• pick colony from BAP1-pKD46 plate from 2013-06-04, grow in 3 ml LB+Amp at 30°C

2013-06-26

• inoculate 50 ml LB + Amp + Ara(0.5%) with 1.1 ml of ON culture
• grow at 30°C to OD=0.73, prepare electrocompetent BAP1-pKD46

2013-06-27

Gel electrophoresis of the PCR amplificate. Lane 1: NEB 2-log ladder; lane 2: 1 µl of PCR amplificate (first PCR)

Gel electrophoresis of the PCR amplificate. Lane 1: NEB 2-log ladder; lane 2: 1 µl of PCR amplificate (second PCR)

Gel electrophoresis of the PCR amplificate. Lane 1: 19 µl of amplificate from first PCR; lane 2: 19 µl of amplificate from second PCR; lane 3: NEB 2-log ladder; lane 4: 1 µl of PCR amplificate (third PCR)

• run PCR of 5 ng pLF03 (1 µl 1:20 dilution of miniPrep from 2013-06-04) with primers IK07 and IK08 using Phusion polymerase (20 µl total volume) on cycler 1 (does not cool down to 4°C) (using hot start):

• no product -> repeat PCR with cycler 2 (fully functional)
• no product -> run 2-step PCR with 4 ng pLF03 (0.2 µl of 21 ng/µl miniPrep from 2013-06-11) with primers IK07 and IK08 using Phusion polymerase (20 µl total volume, using hot start) on cycler 2:

• no product

2013-06-28

Gel electrophoresis of the PCR amplificate. Lane 1: NEB 2-log ladder; lane 2: 1 µl of PCR amplificate

• run PCR of 4 ng pLF03 (0.2 µl of 21 ng/µl miniPrep from 2013-06-11) with primers IK07 and IK08 using Q5 polymerase (20 µl total volume, using hot start) on cycler 2:

• perfect PCR => Phusion is crappy
• run 4 PCRs of 4 ng pLF03 (0.2 µl of 21 ng/µl miniPrep from 2013-06-11) with primers IK07 and IK08 using Q5 polymerase (50 µl total volume, using hot start) on cycler 2:

2013-06-29

Gel electrophoresis of the PCR amplificate. Lane 1: NEB 2-log ladder; lanes 2-5: 1 µl of PCR amplificate

• load 1 µl of PCR product on gel -> specific amplificate
• pool products (also include amplificate from 2013-06-28), purify (only first step, as no DpnI available)

2013-06-30

• inoculate 1 ml LB+Amp with 10 µl of ON culture from 2013-06-25, grow at 30°C

2013-07-01

• finish purification (digest for 5h, dissolve in 50 µl H₂O) -> 1162 ng/µl (performed by Fanny)
• prepare electrocompetent cells from ON culture (add 0.5% arabinose at dilution) (performed by Fanny)
• electroporate fresh electrocompetent cells, 1 aliquot from 2013-06-26 (23 µl DNA each) (performed by Fanny)
• grow in SOC at 30°C for 30 min, transfer to falcons, add 1 ml LB, add IPTG (1 mM), grow at 37°C for 4h
• spin cell down (8000 rcf for 3 min), decant supernatant, resuspend pellet in remaining medium
• plate on Cm+IPTG (2 plates for each sample: 10µl + rest, plates marked F: from -80°C, marked H: newly prepared cells), grow at 37°C

2013-07-02

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG. Lane 1: NEB 2-log; lanes 2, 3: negative control (BAP1-pLF03); lanes 4-9: colony-PCR of F-rest (lanes 8,9: from lawn); lanes 10-13: colony-PCR of H-10µl; lanes 2,4,6,8,10,12: primers IK01+IK02; lanes 3,5,7,9,11,13: primers IK01+IK03 (positive control)

• bacteria lawn on rest-plate from -80°C, possible colonies on other plates (barely visible, may be bubbles)
• pick 2 colonies from F-rest, sample of lawn, 2 colonies from H-10µl, colony-PCR with primers IK01+IK02, IK01+IK03 (positive control) (20µl total volume):

• very evening: colonies on F-10µl, H-rest, H-10µl plates appear -> leave at 37°C ON

2013-07-03

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG.
Lane 1: NEB 2-log
Top: lanes 2-4: negative control (BAP1-pLF03); lanes 5-13: colony-PCR F-10µl
bottom:lanes 2-4: colony-PCR of F-10µl; lanes 5-13: colony-PCR of H-10µl; lanes 2,5,11: primers IK01+IK02; lanes 3,6,12: primers IK01+IK03 (positive control); lanes 4,7,13: primers IK05+IK06 (negative control)

• colonies on F-rest plate appeared (plate left ON at RT)
• pick 4 colonies from F-10µl, 3 colonies from H-10µl, colony-PCR with primers IK01+IK02, IK01+IK03 (positive control), IK05+IK06 (negative control):

• F-10µl 1,2,4 show no negative control -> grow at 37°C
• continue growing plates at 37°C until evening, leave at RT ON

2013-07-04

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG.
Lane 1: NEB 2-log
Top: lanes 2-10: cultures from 2013-07-03; lanes 2,5,8: primers IK01+IK02; lanes 3,6,9: primers IK01+IK03; lanes 4,7,10: primers IK05+IK06; lane 11: BAP1-pLF03 (negative control); lanes 12-13: colonies from F-10µl plate; lanes 11-13: primers IK05+IK06
bottom: lanes 2-13: primers IK05+IK06; lanes 2,3: colonies from F-10µl plate; lanes 4-6: colonies from F-rest plate; lanes 7-10: colonies from H-10µl plate; lanes 11-13: colonies from H-rest plate

• repeat colony-PCR for 3 ambiguous cultures form 2013-07-03 (use 1 µl of culture), pick 14 new colonies (only primers IK05+IK06); 20 µl total volume:

• no positives

2013-07-05

Restriction digest of pKD46. Lane 1: NEB 2-log; lane 2: EcoRI; lane 3: BamHI+NotI; lane 4: PstI+NotI; lane 5: undigested (225 ng (5 ml) loaded)

• digest 450 ng pKD46 (10 µl of 45.5 and 46 ng/µl miniPreps from 2013-06-06) with EcoRI (expected: 4816+1505 bp), BamHI+NotI (expected: 3675+2646 bp), PstI+NotI (expected: 3711+2363+243 bp); 30 µl total volume
• right plasmid
• inoculate 2 x 7 ml LB+Cm+IPTG(1mM) with colonies from F-10µl plate, grow at 30°C

2013-07-06

Restriction digest of BAP1-pKD46 electroporated with pLF03-PCR grown in Cm+IPTG(1mM), miniprepped.
Lane 1: NEB 2-log; lanes 2-5: Konrad's PCR; lane 6: digested with EcoRI; lane 8: undigested

• add Cm (1:3000), IPTG (1:1000) to liquid cultures, grow for 8h at 30°C
• make miniPreps -> 25 ng/µl and 14.4 ng/µl in 27.5 µl
• digest 25 ng/µl miniPrep with EcoRI (use all of miniPrep)
• load digest completely, 10 µl of 14.4 ng/µl miniPrep on gel
• no bands -> nanoDrop crappy, no plasmid
• wrong strain? Streak BAP1 on Cm, grow at 37°C