Team:Heidelberg/Delftibactin/MMCoA

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Revision as of 17:23, 3 October 2013

Methylmalonyl-CoA Pathway. Making the whole thing work.

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Methods:

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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.

2013-06-03

2013-06-04

  • inoculate 4 ml LB+Amp with TOP10-pLF03, grow at 37°C
  • make miniPrep of pLF03 => 97 ng/µl in 27 µl
  • transform BAP1 with pKD46, plate on Amp, grow at 30°C

2013-06-05

  • prepare 50 ml LB + Amp + Arabinose(0.1%), inoculate with BAP1-pKD46
  • prepare 4 ml LB + Amp, inoculate with TOP10-pKD46 (for miniPrep)
  • no colonies with pCP20 => repeat transformation with three times the amount of plasmid (30 µl)
  • BAP1-pKD46 reached OD=0.68 at 22:30 => put in fridge over night

2013-06-06

  • inoculate 50 ml LB + Amp + Arabinose(0.1%) with 500 µl BAP1-pKD46 from ON culture (fridge)
  • at OD=0.49: make glycerol stocks, prepare electrocompetent BAP1-pKD46
  • make miniPrep of TOP10-pKD46 ON culture -> 31 ng/µl
  • low yield of TOP10 miniPrep: make 2 miniPreps of BAP1-pKD46 ON culture -> 45.5 ng/µl and 46.0 ng/µl
  • evening: no colonies with pCP20 -> write Lei Fang (Pfeifer lab) -> their stock is bad, we should get pCP20 somewhere else

2013-06-07

Gel electrophoresis of the PCR amplificate. Left: NEB 2 log DNA ladder; middle: PCR without annealing step; right: full PCR
  • run PCR of 5 ng pLF03 (1 µl 1:20 dilution of miniPrep from 2013-06-04) of with primers ygfG21C1 and ygfG21C2 using Phusion polymerase:
Cycles temperature [°C] Time [s]
1 98 30
30 98 10
66 30
72 90
1 72 450
1 4 inf
  • using hot start at 98°C
  • two PCR samples were run, in one the annealing step at 66°C was left out
  • => no amplificate

2013-06-08

Gel electrophoresis of the PCR amplificate. Left: NEB 2 log DNA ladder; middle: full PCR; right: PCR without 58°C annealing step
  • Looking in more detail at the primers, the parts binding to the template have melting temperatures of 40 and 45°C (the rest is homologous to E. coli genome for recombination)
  • run PCR of 5 ng pLF03 (1 µl 1:20 dilution of miniPrep from 2013-06-04) with primers ygfG21C1 and ygfG21C2 using Phusion polymerase:
Cycles temperature [°C] Time [s]
1 98 30
5 98 10
39 45
72 90
5 98 10
45 45
72 90
20 98 10
58 45
72 90
1 4 inf
  • using hot start at 98°C
  • two PCR samples were run, in one the annealing step at 58°C was left out
  • => no amplificate

2013-06-09

Gel electrophoresis of the PCR amplificate. Left: NEB 2 log DNA ladder; right: PCR product
  • run PCR of 5 ng pLF03 (1 µl 1:20 dilution of miniPrep from 2013-06-04) with primers ygfG21C1 and ygfG21C2 using Phusion polymerase:
Cycles temperature [°C] Time [s]
1 98 30
35 98 10
40 60
72 120
1 72 600
1 4 inf
  • using hot start at 98°C
  • amplificate has 1.3kb -> too short, but specific (one distinct band)

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 (AccA1: 590AS * 3 = 1770 bp, PccB: 530AS * 3 = 1590 bp, 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):
Cycles temperature [°C] Time [s]
1 98 10
5 98 1
45 5
72 120
25 98 1
55 5
72 120
1 72 120
1 4 inf
  • 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):
Cycles temperature [°C] Time [s]
1 94 180
35 94 20
41.5/43 60
72 480
1 72 300
1 4 inf
  • 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):
Cycles temperature [°C] Time [s]
1 95 180
30 95 30
61 30
72 30
1 72 600
1 4 inf
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:
Cycles temperature [°C] Time [s]
1 95 240
30 95 30
61 30
72 30
1 72 600
1 4 inf
  • 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:
Cycles temperature [°C] Time [s]
1 95 600
30 95 240
64 30
72 60
1 72 600
1 4 inf
Cycles temperature [°C] Time [s]
1 95 600
12 95 240
66°C ↓0.5°C 30
72 60
18 95 240
60°C 30
72 60
1 72 600
1 4 inf
  • 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
Cycles temperature [°C] Time [s]
1 98 10
5 98 1
45 5
72 130
25 98 1
55 5
72 120
1 72 120
1 4 inf
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)
Cycles temperature [°C] Time [s]
1 95 300
30 95 60
62 30
72 60
1 72 600
1 4 inf
  • 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:
Cycles temperature [°C] Time [s]
1 95 300
12 95 60
68 ↓0.5°C 30
72 120 (IK01+IK02; IK01+IK03;IK05+IK06) / 240 (IK01+IK06)
18 95 60
62 30
72 120 (IK01+IK02; IK01+IK03;IK05+IK06) / 240 (IK01+IK06)
1 72 600
1 4 inf
  • 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):
Cycles temperature [°C] Time [s]
1 98 30
30 98 5
66 30
72 150
1 72 600
1 4 inf
  • 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:
Cycles temperature [°C] Time [s]
1 98 30
30 98 5
72 150
1 72 600
1 4 inf
  • 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:
Cycles temperature [°C] Time [s]
1 98 30
30 98 5
66 30
72 150
1 72 1800 (30 min)
1 4 inf
  • 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:
Cycles temperature [°C] Time [s]
1 98 30
45 98 5
66 30
72 150
1 72 1800 (30 min)
1 4 inf

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):
Cycles temperature [°C] Time [s]
1 95 300
12 95 60
68 ↓0.5°C 30
72 120
18 95 60
62 30
72 120
1 72 600
1 4 inf
  • 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):
Cycles temperature [°C] Time [s]
1 95 300
12 95 60
68 ↓0.5°C 30
72 120
18 95 60
62 30
72 120
1 72 600
1 4 inf
  • 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:
Cycles temperature [°C] Time [s]
1 95 300
12 95 60
68 ↓0.5°C 30
72 120
18 95 60
62 30
72 120
1 72 600
1 4 inf
  • 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

2013-07-08

gel electrophoresis of PCR of pLF03 with primers IK07 and IK08 using Q5 polymerase. Product from one PCR tube was split to two neighboring lanes.
1 µl of gel-extracted PCR amplificate was loaded. Lane 1: NEB 2-log; lane 2: amplificate extracted by Dominik, lane 3: amplificate extracted by Ilia
  • no colonies of BAP1 on Cm
  • repeat PCR of pLF03:
  • 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):
Cycles temperature [°C] Time [s]
1 98 30
45 98 5
66 30
72 150
1 72 1800 (30 min)
1 4 inf
  • extract amplificate from gel (4 lanes pooled, extracted by Dominik -> 119 ng/µl; 4 lanes pooled, extracted by Ilia -> 111.5 ng/µl)
  • load 1 µl of each extract on gel

2013-07-09

  • electroporate electrocompetent BAP1-pKD46 with 1µl, 5µl, 25µl DNA
  • resuspend in 1 ml SOC + IPTG (1mM) + Ara (0.1%)
  • grow for 90 min at 37°C, 400 rpm
  • spin cells down, plate 10µl, rest of each sample on Cm+IPTG

2013-07-10

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG with primers IK05+IK06. Lane 1: NEB 2-log; lanes 2, 13: BAP1-pLF03 (control); lanes 3-12: colony-PCRs of large colonies from 25µl/10µl plate. Lanes 2-12: OneTaq; lane 13: iTaq
  • on all plates: large + small colonies
  • small colonies too small to pick => pick 10 large colonies from 25µl DNA / 10 µl bacteria plate, run colony-PCR with primers IK05+IK06 (Taq, 20 µl total volume):
Cycles temperature [°C] Time [s]
1 95 300
12 95 60
68 ↓0.5°C 30
72 120
18 95 60
62 30
72 120
1 72 600
1 4 inf
  • no integration
  • continue growing plates at 37°C
Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG.
Top: lane 1: NEB 2-log; lanes 2,4,6,8,10,12: primers IK05+IK06; lanes 3,5,7,9,11,13: primers IK01+IK03 (positive control); lanes 2,3: BAP1-pLF03 (control); lanes 4-7: colonies from 1µl/10µl plate; lanes 8-11: 1µl/rest; lanes 12-13: 5µl/10µl
Bottom: lane 11: NEB 2-log; lanes 1,3,5,7,9: primers IK05+IK06; lanes 2,4,6,8,10: primers IK01+IK03 (positive control); lanes 1-2: 5µl/10µl; lanes 3-6: 5µl/rest; lanes 7-8: 25µl/10µl; lanes 9-10: 25µl/rest
  • when small colonies large enough: pick, run colony-PCR (OneTaq, 20µl total volume):
Cycles temperature [°C] Time [s]
1 95 300
12 95 60
68 ↓0.5°C 30
72 120
23 95 60
62 30
72 120
1 72 600
1 4 inf
  • 1st colony from 5µl/rest plate (marked 7) seems promising => grow at 37°C in 1 ml LB

2013-07-11

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG and then in LB at 37°C. Lanes 1,4: primers IK01+IK03 (positive control); lanes 2,5: primers IK01+IK02; lanes 3,6: primers IK05+IK06; lanes 1-3: BAP1-pLF03 (control); lanes 4-6: colony 7; lane 7: NEB 2-log
  • run full colony-PCR of liquid culture (OneTaq, 20 µl total volume, use 1 µl of culture):
Cycles temperature [°C] Time [s]
1 95 300
12 95 60
68 ↓0.5°C 30
72 120
18 95 60
62 30
72 120
1 72 600
1 4 inf
  • no integration
  • 3rd population of colonies appeared on plates (stored at RT): very small, probably satellites
Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG and then in LB at 37°C with primers IK07+IK08. Lane 1: NEB 2-log; lane 2: BAP1-pLF03 (control); lane 3: large colony picked from 25µl/10µl plate; lane 4: colony marked 1 from 1µl/10µl plate; lane 5: colony marked 6 from 5µl/10µl plate; lane 6: colony marked 7 from 5µl/rest plate.
  • run colony-PCR of liquid with primers IK07+IK08 (test for integration in wrong place) (OneTaq, 20µl total volume, use 1 µl liquid culture):
Cycles temperature [°C] Time [s]
1 95 300
30 95 60
66 30
72 600
1 72 600
1 12 inf
  • no integration at all
Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG with primers IK05+IK06.
Top: lane 1: NEB 2-log; lane 2: BAP1-pLF03(control); lanes 3-9: colonies from 1µl/10µl plate; lanes 10-13: colonies from 1µl/rest plate
Bottom: lane 1: NEB 2-log; lanes 2-10: colonies from 1µl/rest plate (lane 5: swiped 3rd population of extremely small colonies)
  • pick 20 new colonies, run colony-PCR with primers IK05+IK06 (iTaq, 20 µl total volume):
Cycles temperature [°C] Time [s]
1 95 300
12 95 60
68 ↓0.5°C 30
72 120
23 95 60
62 30
72 120
1 72 600
1 4 inf
  • unspecific bands (also present in colony-PCR from 2013-07-10)
  • unspecific bands not present in BAP1-pLF03 PCR (control) and bottom lane 5 (satellite colonies) => not due to iTaq
  • assume: some amount of satellites was picked together with colonies from plates -> give band at 500 bp; partially amplified ygfG fragments prime integrated insert
  • do multiple sequence alignment of ygfG, pccB, accA1, and catR using ClustalO => long enough stretches of partially identical sequences present
  • add 1mM IPTG to cultures with colonies from 1µl/10µl plate picked today (grew in 500 µl LB at 37°C for several hours), after 2h at 37°C: streak on Cm+IPTG
  • pick 10 colonies each from 1µl/10µl and 1µl/rest plates, transfer to new Cm+IPTG plate, grow at 37°C
  • pick 1 colony from 1µl/10µl plate, inoculate 1 ml LB + Cm + IPTG (1mM)

2013-07-12

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG with primers IK01+IK02.
Top: lane 1: NEB 2-log; lane 2: BAP1-pLF03(control); lanes 3-12: colonies liquid culture that were plated on Cm+IPTG; lane 13: colony from 1µl/10µl plate that was transferred to new Cm+IPTG plate
Bottom: lane 1: NEB 2-log; lanes 2-6: colonies from 1µl/10µl plate that were transferred to new Cm+IPTG plate; lanes 7-12: colonies from 1µl/rest plate that were transferred to new Cm+IPTG plate; lane 13: liquid culture (Cm+IPTG(1mM)) from 1µl/10µl plate
  • colonies grew slowly
  • run colony-PCR with primers IK01+IK02 (iTaq, 20 µl total volume):
Cycles temperature [°C] Time [s]
1 95 300
12 95 60
68 ↓0.5°C 30
72 120
23 95 60
62 30
72 120
1 72 600
1 4 inf
  • no bands
Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG with primers IK05+IK06. Lane 1: NEB 2-log; lane 2: BAP1-pLF03 (control); lanes 3-4: colony-PCRs (from colonies marked 1 and 2)
  • run colony-PCR with primers IK05+IK06 of 2 colonies from liquid culture that were transferred to Cm + IPTG plate (iTaq, 20µl total volume):
Cycles temperature [°C] Time [s]
1 95 300
12 95 60
68 ↓0.5°C 30
72 120
23 95 60
62 30
72 120
1 72 600
1 4 inf
  • band at 1.2 kb is now specific -> WTF?!?

2013-07-13

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2) with primers IK05+IK06. Lane 1: NEB 2-log; lanes 2,4,6,8,10,12: BAP1-pLF03 (control); lanes 3,5,7,9,11,13: colony-PCR; lanes 2,3: annealing at 63°C; lanes 4,5: 64°C; lanes 6,7: 65°C; lanes 8,9: 66°C; lanes 10,11: 67°C; lanes 12,13: 68°C.
  • need to determine whether 1.2 kb band is specific: run gradient PCR of colony 2 (iTaq, 20 µl total volume) with primers IK05+IK06:
Cycles temperature [°C] Time [s]
1 95 300
35 95 60
63-68 (ΔT = 1) 30
72 120
1 72 600
1 4 inf
  • intensity of 1.2kb band decreases, intensity of 0.5 kb band (control) does not -> 1.2 kb band is unspecific product
  • check for presence of genomically integrated insert: run colony-PCR of colony 2 with primers IK07+IK08 (iTaq, 20 µl total volume):
Cycles temperature [°C] Time [s]
1 95 300
35 95 60
66 30
72 600
1 72 600
1 12 inf
  • transfer some of colony 2 on Amp (check for presence of pLF03 as a whole), grow at 37°C
  • transfer some of colony 2 to liquid culture: LB + Cm + IPTG (1mM), grow at 37°C

2013-07-14

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2) with primers IK07+IK08. Lane 1: NEB 2-log; lane 2: BAP1-pLF03 (control); lane 3: colony-PCR
  • bacteria grew on Amp => 3 possibilities:
    • Amp plates not selective
    • bacteria have complete pLF03 plasmid
    • bacteria still have pKD46, although grown at 37°C
  • plate BAP1, BAP1-pKD46 on Amp, grow at 37°C
  • colony-PCR did not work for the control -> repeat with OneTaq (use 1 µl of liquid culture)
  • no amplificate for colony: possibly, Taq has problems amplifying 4.8 kb from genomic DNA
Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2) and grown in liquid culture with Cm+IPTG(1mM). Lane 3: NEB 2-log; lanes 1,4: BAP1-pLF03 (control); lanes 2,5: 1 µl of liquid culture; lanes 1,2: primers IK07+IK08; lanes 4,5: primers IK01+IK06
  • run colony-PCR with primers IK01+IK06 (OneTaq, 20 µl total volume, use 1 µl of liquid culture):
Cycles temperature [°C] Time [s]
1 95 300
12 95 60
68 ↓0.5°C 30
72 240
23 95 60
62 30
72 240
1 72 600
1 4 inf
  • control shows bright band where expected, colony does not => something is integrated, unknown what

2013-07-15

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2) and grown in liquid culture with Cm+IPTG(1mM) with primers IK07+IK08. Lane 1: NEB 2-log; lane 2: BAP1-pLF03 (control); lane 3: 1 µl of liquid culture.
Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2) with primers IK07+IK08. Lane 1: NEB 2-log; lane 2: BAP1-pLF03 (control); lane 3: 1 µl of liquid culture.
  • BAP1 did not grow on Amp, BAP1-pKD46 did grow on Amp at 37°C => no discrimination between pLF03 and pKD46 possible
  • Taq might have problems amplifying 4.8 kb from genomic DNA => use Phusion Flash (primers IK07+IK08, 20 µl total volume, use 1 µl of liquid culture, old BioRad cycler):
Cycles temperature [°C] Time [s]
1 98 10
35 98 1
66 5
72 100
1 72 600
1 12 inf
  • control did not work -> repeat in new BioRad cycler (pick from plate)
  • same result

2013-07-18

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2). Lane 1: NEB 2-log; lanes 2,4,6: BAP1-pLF03 (control); lanes 3,5,7: colony-PCR. Lanes 2,3: primers IK24+IK25; lanes 4,5: primers IK07+IK25; lanes 6,7: primers IK01+IK25
Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2). Lane 1: NEB 2-log; lanes 2,4,6: BAP1-pLF03 (control); lanes 3,5,7: colony-PCR. Lanes 2,3: primers IK24+IK25; lanes 4,5: primers IK07+IK25; lanes 6,7: primers IK01+IK25
  • run colony-PCR with primers IK24, IK25 (OneTaq, 20 µl total volume):
Cycles temperature [°C] Time [s]
1 95 300
12 95 60
68 ↓0.5°C 30
72 180
23 95 60
62 30
72 180
1 72 600
1 12 inf
  • 1.2 kb band with primers IK01+IK25 in control => makes no sense, repeat
  • same result
  • transfer BAP1-pLF03 to new Cm+IPTG plate, colony 2 to LB w/o antibiotics, grow at 37°c#

2013-07-22

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2). Lane 1: NEB 2-log; lanes 2,4: BAP1-pLF03 (control); lanes 3,5: colony-PCR. Lanes 2,3: primers IK01+IK03; lanes 4,5: primers RB43+RB44
Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2) with primers RB43+RB44. Lane 1: NEB 2-log; lane 2: BAP1-pLF03 (control); lane 3: colony-PCR; lane 4: streaked BAP1 by Konrad
  • no positive PCR of colony yet: run colony-PCR with primers IK01+IK03 (iTaq, 20 ml total volume):
Cycles temperature [°C] Time [s]
1 95 300
12 95 60
68 ↓0.5°C 30
72 180
23 95 60
62 30
72 180
1 72 600
1 12 inf
  • primers RB43+RB44 (against sfp) iTaq, 20 µl total volume:
Cycles temperature [°C] Time [s]
1 95 300
35 95 60
55 30
72 180
1 12 inf
  • unspecific bands in colony, no sfp bands at all
  • re-run sfp PCR (primers RB43+RB44), include streaked BAP1 by Konrad
  • sfp present in BAP1, not present in BAP1-pLF03, colony
  • makes no sense, Konrad streaked his cells from the same batch of competent cells
  • transfer BAP1, BAP1-pLF03, colony to liquid culture LB, grow at 37°C

2013-07-23

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2) and grown in liquid culture with primers RB43+RB44. Lane 1: NEB 2-log; lane 2: BAP1-pLF03 (control); lane 3: colony-PCR; lane 4: streaked BAP1 by Konrad, lane 5: BAP1-pMM64-pMM65 from 2013-06-11
  • run colony-PCR with primers RB43+RB44 (iTaq, 20 µl total volume, use 1 µl of liquid culture)
Cycles temperature [°C] Time [s]
1 95 300
35 95 60
55 30
72 180
1 72 600
1 12 inf
  • no sfp present

== Goal ==

Vector map of the pIK1 plasmid.
Vector map of the pIK2 plasmid.

Back-up plan in case the genomic integration does not work. Two plasmids are to be constructed, pIK1 (containing BBa_I746200, pccB-accA, and sfp) and pIK2 (containing pccB-accA and sfp) in the pSB3C5 backbone. 6 fragments are to be amplified:

fragment ID source primers size [kb]
f1 BBa_J04450 in pSB3C5 IK32+IK33 3
f2 BBa_I746200 IK26+IK27 2.3
f3 pccB-accA in pLF03 IK28+IK29 3.4
f4 sfp in BAP1 IK30+IK31 0.7
f5 BBa_J04450 in pSB3C5 IK32+IK35 3
f6 pccB-accA in pLF03 IK34+IK29 3.4

2013-07-22

  • transform BBa_I746200 in pSB1AK3 (Spring 2012 Distribution Plate 2 Well 15E) in TOP10, plate on Amp
  • transform BBa_J04450 in pSB3C5 (Spring 2012 Distribution Plate 1 Well 3C) in TOP10, plate on Cm

2013-07-23

  • pick TOP10-BBa_I746200 colony, inoculate LB+Kan, grow at 37°C
  • pick TOP10-BBa_J04450 colony, inoculate LB+Cm, grow at 37°C
  • after 12 hours: add new antibiotics to medium, continue growing at 37°C

2013-07-24

2013-07-25

File:Methylmalonyl-PCR2013-07-25 1.png
Lane 1: NEB 2-log; lane 3: f1; lane 4: f2; lane 5: f3; lane 6: f4; lane 7: f5; lane 8: f6
  • run PCRs (using Q5, 20 µl total volume) using 4 ng of each template (pLF03 miniPrep from 2013-06-11, 21 ng/µl), sfp from Konrad's BAP1 plate:
Cycles temperature [°C] Time [s]
1 98 30 (f1,f2,f3,f5,f6) / 300 (f4)
35 98 5
66 (f1,f2) / 60 (f4) / 61 (f3,f5,f6) 10
72 120 (f1,f2,f3,f5,f6) / 30(f4)
1 72 600
1 12 inf
  • did not manage to get f3, f4, f6
  • repeat: add 1 µl DMSO to reactions:
File:Methylmalonyl-PCR2013-07-25 2.png
Lane 1: NEB 2-log; lane 2: f3; lane 3: f4 with lots of bacteria from new plate; lane 4: f4 with lots of bacteria from old plate; lane 5: f4 with little bacteria from new plate; lane 6: f6
Cycles temperature [°C] Time [s]
1 98 30
35 98 5
56 (f4) / 57 (f3,f6) 30
72 150 (f3,f6) / 45(f4)
1 72 600
1 10 inf
File:Methylmalonyl-PCR2013-07-25 3.png
Lane 1: NEB 2-log; lane 2: f3; lane 3: f6
  • use pLF03 miniPrep from 2013-06-11, 27 ng/µl, repeat for f3, f6 (with 1 µl DMSO):
Cycles temperature [°C] Time [s]
1 98 30
12 98 10
62 ↓0.5°C 10
72 150
23 98 10
58 10
72 150
1 72 600
1 10 inf

2013-07-26

File:Methylmalonyl-PCR2013-07-26.png
Lane 1: NEB 2-log; lane 2: f3 with annealing; lane 3: f6 with annealing; lane 4: f6 2-step; lane 5: f3 2-step
  • run PCR of f3, f6 with Phusion Flash (using pLF03 miniPrep from 2013-06-11, 21 ng/µl), without DMSO:
Cycles temperature [°C] Time [s]
1 98 10
35 98 1
65 5
72 75
1 72 600
1 10 inf
Cycles temperature [°C] Time [s]
1 98 10
5 98 1
65 5
72 75
30 98 1
72 75
1 72 600
1 10 inf
  • inoculate 10 ml LB+Amp with TOP10-pLF03, grow for 36h at 37°C, add Amp every 12 hours

2013-07-28

  • make miniPrep -> very low yield (ca. 1-2 ng/µl)
  • inoculate 6 ml LB+Amp with TOP10-pLF03, grow at 37°C

2013-07-29

  • inoculate 10 ml 2xYT+Amp with TOP10-pKD46, grow at 30°C

2013-07-31

miniPrep of pKD46. Lane 1: NEB 2-log (5 µl), lane 2: 1 µl of miniPrep
  • miniPrep of pKD46 -> ca. 10 ng/µl

2013-08-03

  • transform BAP1 with 5µl of pKD46 miniPrep form 2013-07-31, plate on Amp, grow at 30°C

2013-07-29

1 µl of pLF03 miniPrep from 2013-06-04
  • make miniPrep -> very low yield (1-2 ng/µl)
  • load 1µl of 97ng/µl pLF03 miniPrep from 2013-06-04 on gel -> ca. 20 ng/µl
  • inoculate 50 ml LB+Amp with TOP10-pLF03, grow at 37°C

2013-07-30

1 µl of pLF03 midiPrep from 2013-07-30
  • make midiPrep -> 1352 ng/µl in 66.5 µl
  • medium was contaminated (also check with Fanny) -> low yields of previous miniPreps

2013-07-31

  • send 20 µl of 1:15 dilution of midiPrep to sequencing with primers IK07, IK36, IK37: File:Heidelberg PLF03 seq 2013-07-31.zip
  • perform multiple sequence alignment against assumed pLF03 sequence: IK07, IK36, IK37
  • deletion at the end of accA2 -> primer can not bind

2013-08-05

  • run colony-PCR against sfp on BAP1-pKD46 with primers RB43+RB44 (iTaq, 20 µl total volume):
Colony-PCR of BAP1-pKD46 with primers RB43+RB44. Lane 1: sfp, lane 2: NEB 2-log
Cycles temperature [°C] Time [s]
1 95 300
35 95 60
55 30
72 180
1 72 600
1 10 inf
  • inoculate 4 ml 2xYT+Amp with BAP1-pKD46, grow at 30°C

2013-08-06

Lane 1: NEB 2-log; lane 3: colony-PCR with primers RB43+RB44
  • inoculate 50 ml 2xYT+Amp+Ara(0.5%) with BAP1-pKD46, grow at 30°C until OD=0.71
  • run colony-PCR with primers RB43+RB44 from 50 ml culture:
Cycles temperature [°C] Time [s]
1 95 300
35 95 60
55 30
72 180
1 72 600
1 10 inf

2013-08-08

  • electroporate BAP1-pKD46 with 500 ng DNA (5 µl of PCR amplificate from 2013-07-08)
  • grow in SOC + IPTG(1mM) + Arabinose (0.5%) at 37°C, 400 rpm for 3h
  • spin cells down (3 min at 8000 rcf)
  • plate 10 µl, rest on Cm, grow at 37°C

2013-08-05

Lane 1: NEB 2-log; lane 2: f3; lane 3: f6
  • received constructs from Matthew Mattozzi, Boston: pET21c-pcc-acca-mcce in NEB Turbo (Ampr), pWH8: mcc-pcs-pcc-acc-mmce in NEB Turbo (Kanr), E. coli MM17: MG1655 lambda(DE3) d(lacY) d(E14) mcr-pcs-pcc-acc::KanR integrated at phage site HK022 (Kanr)
  • plates contaminated with Drosophila larvae
  • re-streak on fresh plates, run colony-PCR for f3, f6 from pET21c-pcc-acca-mcce (Q5, 20 µl total volume):
Cycles temperature [°C] Time [s]
1 98 120
35 98 5
61 10
72 120
1 72 600
1 10 inf
  • no bands

2013-08-06

Lane 1: NEB 2-log; lane 2: f3; lane 3: f6
  • run colony-PCR from pET21c-pcc-acca-mcce grown ON (Q5, 20 µl total volume) for fragments f3, f6:
Cycles temperature [°C] Time [s]
1 98 60
35 98 5
61 10
72 120
1 72 600
1 10 inf
  • inoculate 5 ml 2xYT+Amp with colony from which colony-PCR was run, grow at 37°C
  • no fragment for f3, re-run PCR (1 µl of liquid culture, 20 µl total, Phusion Flash):
Lane 1: NEB 2-log; lane 2: f3; lane 3: from genomic integration; lane 4: 2 µl of pET21c-pcc-acc-mmce miniPrep.
Cycles temperature [°C] Time [s]
1 98 60
35 98 1
65 5
72 60
1 72 600
1 10 inf
  • make miniPrep of liquid culture
  • no PCR product, miniPrep runs above 10 kb supercoiled (whole plasmid is expected to have 9 kb) (miniPrep has ca. 10 ng/µl)
  • re-run PCR for f3 from miniPrep (Q5, 0.2 µl DNA, 20 µl total volume):
Cycles temperature [°C] Time [s]
1 98 60
35 98 5
61 10
72 120
1 72 600
1 10 inf

2013-08-07

Lane 1: NEB 2-log; lane 2: f3; lane 3: BamHI digest of pET21c-pcc-acc-mcce; lane 4: 1 kb plus ladder.
  • need to verify plasmid identity: linearize with BamHI (unique restriction site in both pET21c... and pWH8, use 5 µl of miniPrep, 30 µl total volume)
  • linearized pET21c... plasmid runs at 9 kb -> right
  • PCR of f3 worked -> hypothesis: Gibson overhang against permeability device bound to E. coli genome in colony-PCRs, as permeability device is mutated version of FepA, which is present in E. coli
  • gel-purify all fragments
  • f1: 54.2 ng/µl, f2: 64.4 ng/µl, f3: 24.4 ng/µl, f4: 14 ng/µl, f5: 47.6 ng/µl, f6: 25.2 ng/µl; all in 30 µl
  • perform Gibson assembly:
    • pIK1: 0.8 µl f1, 1.6 µl f2, 5 µl f3, 2 µl f4, 0.4 µl H2O, 10 µl NEB Gibson MM
    • pIK2: 1 µl f5, 6 µl f6, 3 µl f4
    • incubate at 50°C for 1h
  • transform TOP10: 2 transformations per plasmid: 5 µl of Gibson assembly, 5 µl of 1:4 diluted Gibson assembly
    • grow in 2xYT at 37°C, plate on Cm, grow at 37°C

2013-08-08

  • pIK1: several colonies, some of them pink -> pick 10 white, grow in 2xYT+Cm at 37°C
  • pIK2: 2 populations of colonies: large and small, several large colonies are pink -> pick 5 white large, 5 white small colonies, grow in 3 ml 2xYT+Cm at 37°C

2013-08-09

Lane 1: NEB 2-log; lanes 2-10: pIK1 miniPreps digested with BamHI+HindIII; lanes 11-13: large colonies of pIK2 miniPreps digested with BamHI+HindIII
Lane 1: NEB 2-log; lanes 2-3: large colonies of pIK2 miniPreps digested with BamHI+HindIII; lanes 4-8: small colonies of pIK2 miniPreps digested with BamHI+HindIII; lanes 9-13: Flo's PCR
  • colony 8 of pIK1 did not grow
  • make miniPreps of 2 ml everything else (2 ml each) -> ca. 62 - 600 ng/µl
  • miniPrep yields quite high, but consistent: pACYC with p15A origin (4 kb) yields 0.6 µg DNA / ml culture in LB, culture in 2xYT/TB yields 2-5 times more cells (according to QiaGen) => ca. 10-15 µg expected for 9 kb plasmid
  • digest with BamHI+HindIII (20 µl total volume, 0.5 µl enzyme, 1 µl DNA [except for sample with 62 ng/µl, there 2 µl DNA was used])
  • expected fragments: 2151 bp + 7265 bp for pIK1, 2151 bp + 5029 bp for pIK2
  • send pIK1.2 and pIK2.2 to sequencing with primers VF2 and VR

2013-08-12

Lane 1: NEB 2-log; lanes 2-4: Colony-PCRs with primers IK01+IK02
  • no colonies on 10 µl plate
  • 2 large colonies, 1 small colony on rest plate
  • run colony-PCR with primers IK01+IK02:
Cycles temperature [°C] Time [s]
1 95 300
12 95 60
68 ↓0.5°C 30
72 120
23 95 60
62 30
72 120
1 72 600
1 12 inf
  • genomic integration failed

== 2013-08-12 ==

  • sequences arrived: File:PIK1.2-pIK2.2 seq 2013-08-12.zip
  • perform multiple sequence alignment against assumed sequences: pIK1.2 with VF2, pIK1.2 with VR, pIK2.2 with VF2, pIK2.2 with VR
  • pIK1.2 looks good
  • part of RBS missing in pIK2.2 -> makes no sense, as part of binding part of IK35, with which the backbone was amplified
  • send pIK2.6 to sequencing with primers VF2 and VR, also sequence pIK1.2 with primer IK25 to find out whether RBS is also missing there

    • 2013-08-13

    2013-08-14

    2013-08-19

    • received BL21(DE3) from Gruss Lab, ZMBH
    • grow in LB to OD=0.53, prepare competent cells
    • co-transform with pIK1.2(0.2 µl = 120 ng)+pMM64(1 µl = 15 ng), pIK2.6(0.2 µl = 60 ng)+pMM64(1 µl = 15 ng), plate on Cm+Amp+IPTG, grow at 37°C

    2013-08-20

    • all colonies white
    • pick 1 colony each, inoculate LB+Cm+Amp, grow at 37°C until cultures are opaque, add IPTG to 1mM, grow at 30°C
    • no visible indigoidine production
    • need to clone into a different backbone in order to be able to cotransform with pRB21
    • inoculate LB+Kan with TOP10-pSB3K3-BBa_J04450, grow at 37°C

    2013-08-21

    Lane 1: NEB 2-log; lanes 2-3: pSB3K3-BBa_J04450 digested with EcoRI+SpeI; lane 4: pIK1.2 digested with EcoRI+SpeI; lane 5: pIK2.6 digested with EcoRI+SpeI
    • prepare glycerol stock of TOP10-pSB3K3-BBa_J04450
    • perform miniPrep -> 10.1 ng/µl in 38 µl
    • digest with EcoRI+SpeI: 380 ng of pSB3K3-BBa_J04450: use everything (50 µl total volume, 2x0.5 µl enzyme, expect 2.7kb & 1kb), 1200 ng of pIK1.2 (2 µl of miniPrep from 2013-08-09, 20 µl total volume, 2x0.5 µl enzyme, expect 6.7kb & 2.7kb), 900 ng of pIK2.6 (3 µl of miniPrep from 2013-08-09, 20 µl total volume, 2x0.5 µl enzyme, expect 4.4kb & 2.7kb)
    • pSB3K3-BBa_J04450 looks as though it was not cut: inoculate 50 ml TB+Kan with TOP10-pSB3K3-BBa_J04450, grow at 37°C
    • as control for indigoidine production from pMM64: co-transform BL21(DE3) with pMM64(1 µl = 15 ng)+pMM65(0.2 µl = 13 ng), plate on Amp+Kan+IPTG

    2013-08-22

    Lane 1: pSB3K3-BBa_J04450 digested with EcoRI+SpeI, lane 2: NEB 2-log
    Lane 1: NEB 2-log; lanes 2-9: pRB* plasmids; lane 10: 1 µl of pSB3K3-BBa_J04450 midiPrep
    • no colonies of BL21(DE3)-pMM64-pMM65: co-transform BL21(DE3) with pMM64(1 µl = 15 ng)+pMM65(1 µl = 75 ng), plate on Amp+Kan+IPTG
    • prepare midiPrep of pSB3K3-BBa_J04450: 2952.1 ng/µl
    • digest 0.4 µl of midiPrep with EcoRI+SpeI (20 µl total volume, 2x0.5 µl enzyme)
    • band too weak: 2 possibilities:
      1. concentration measurement wrong
      2. mispipeted
    • => load 1 µl of midiPrep on gel -> concentration measurement about right
    • large pSB3K3 fragment over 3 kb, consistent with miniPrep from 2013-08-21 -> 2.7 kb expected, sequence wrong on parts.igem.org
    • gel-purify pSB3K3, 6 kb fragment of pIK1, 4 kb fragment of pIK2
    • 18 µl of pSB3K3 (not measured), 12.3 ng/µl pIK1 in 21 µl; 7.9 ng/µl pIK2 in 21 µl
    • ligate at RT for 1h:
    what µl
    pSB3K3 9
    insert 8
    T4 ligase 1 µl
    T4 ligase buffer 2 µl
    • heat-inactivate: 75°C for 5 min
    • transform 10 µl of each ligation into TOP10, plate on Kan, grow at 37°C (pSB3K3+pIK2 = pIK6)

    2013-08-23

    Colony-PCR of TOP10 transformed with pSB3K3 ligated with pIK1/pIK2 and primers RB43+VR. Lane 1: NEB 2-log; lanes 2-6: pIK1; lanes 7-11: pIK2
    Colony-PCR of TOP10 transformed with pSB3K3 ligated with pIK2 and primers RB43+VR. Lane 1: NEB 2-log; lanes 2-11: pIK2
    • pick 5 colonies each, run colony-PCR with primers RB43+VR (expected: 1kb, using iTaq, 20 µl total volume):
    Cycles temperature [°C] Time [s]
    1 95 300
    35 95 30
    54 30
    72 90
    1 72 600
    1 10 inf
    • no amplificate
    • have another look at sequences (motivated by DelRest results): 1 bp missing in pIK1.2: frame shift in permeability device => send pIK1.3 to sequencing with primers VF2, VR, IK25
    • pick 30 colonies of pSB3K3-pIK2, run colony-PCR with primers RB43+VR (pool 3 colonies in 1 PCR reaction, iTaq, 20 µl total volume)
    • no positives => ligation did not work, most likely due to low DNA concentrations
    • need to repeat: inoculate 2x5 ml 2xYT+Cm with TOP10-pIK2.6

    2013-08-24

    Lane 1: NEB 2-log; lane 3: pSB3K3 digested with EcoRI+SpeI; lane 5: pIK2.6 digested with EcoRI+SpeI
    • make miniPrep of pIK2.6: 97 ng/µl in 37.5 µl
    • digest 3 µg pSB3K3 (1 µl of midiPrep from 2013-08-22), 1.7 µg pIK2.6 (17 µl of miniPrep) with EcoRI+SpeI (20 µl total volume, 0.5 µl of each enzyme)
    • treat pSB3K3 digest with antarctic phosphatase (37°C, 60 min)
    • run gel, gel-purify -> 18 µl pSB3K3 (not measured), 15.0 ng/µl pIK2.6 in 20.5 µl
    • ligate at RT for 1h:
    what µl
    pSB3K3 9
    pIK2.6 8
    T4 ligase 1 µl
    T4 ligase buffer 2 µl
    • heat-inactivate: 75°C for 5 min
    • transform 10 µl of ligation into TOP10, plate on Kan, grow at 37°C

    2013-08-25

    Colony-PCR of TOP10 transformed with pSB3K3 ligated with pIK2 and primers RB43+VR. Lane 1: NEB 2-log; lanes 2-11: pIK2
    • pick 10 colonies, run colony-PCR with primers RB43+VR (expected: 1kb, using iTaq, 20 µl total volume):
    Cycles temperature [°C] Time [s]
    1 95 300
    35 95 30
    54 30
    72 90
    1 72 600
    1 10 inf
    Colony-PCR of TOP10 transformed with pSB3K3 ligated with pIK2 and primers VF2+IK25. Lane 1: NEB 2-log; lanes 2-11: pIK2
    Cycles temperature [°C] Time [s]
    1 95 300
    35 95 30
    54 30
    72 90
    1 72 600
    1 10 inf
    • definite product, but at 1.3 kb => pSB3K3 was longer than expected in digest, might be a result from that
    • unspecific bands probably due to melting temperature of IK25 being at 67°C
    • inoculate 2xYT+Kan, grow at 37°C
    • pSB3K3 used was from plate 5 => transform TOP10 with pSB3K3 from plate 2, well 6 F

    Retrieved from "http://2013.igem.org/Team:Heidelberg/Delftibactin/MMCoA"