Team:Heidelberg/Delftibactin/MMCoA

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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 ([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):
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, [http://www.ncbi.nlm.nih.gov/nuccore/AF113605 pccB], [http://www.ncbi.nlm.nih.gov/nuccore/AF113603 accA1], and [http://www.ncbi.nlm.nih.gov/nuccore/AY048742 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

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

29-07-2013

Restriction digest of fragment FS_02 to FS_03 (5.3 kb; 27-07-2013; II) with BglII

Test restriction digest (29.07); run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for about 3 h

what µL
FS_02 to FS_03 (27-07-2013; II) 15
BglII 1
Buffer 3.1 2
dd H2O 2
Expected fragment lengths [bp] 2146, 1862, 1306


Results:

  • Restriction digest shows the expected product sizes
  • indicator for correct amplicon but to be sure, PCR product will be prepared for single read sequencing by GATC

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

07-08-2013

Sequencing Results

We send the following samples (amplified fragments of D. acidovorans DSM-39 and backbone pSB4K5 from the partsregistry) to GATC for sequencing:

  • AE (FS_02-FS_03) with Primer FS_02
  • AE (FS_02-FS_03) with Primer FS_03

Alignment of DelAE(FS_02 to FS_03) with Primer FS_02

Alignment of DelAE(FS_02 to FS_03) with Primer FS_03

08-08-2013

Amplification from FS_02 to FS_03; 5.3kb

Amplifcation of DelAE and DelEF and DelOP; run at 100 V, 0.8 % gel (TAE)(07.08)
Amplifcation of DelAE and DelEF and DelOP; run at 100 V, 0.8 % gel (TAE)(07.08)
Reaction
what µL
D. acidovorans SPH-1 1
FS_02: (1/10) 4
FS_03: (1/10) 4
Phusion flash Master Mix 10
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
58 5
72 1:30
1 72 7min
1 12 inf

Results:

  • Amplification of DelAE was repeated successfully with the new strain SPH-1
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • if fragment is used in Gibson assembly the amplification has to be repeated to increase the amount of product

12-08-2013

Amplification from FS_02 to FS_05; 11.2 kb

Amplification of DelAF using gradient PCR, Amplification of DelOP 72°C 2-step; run at 135 V, 0.8 % gel (TAE)(11.08)
Amplification of DelAF using gradient PCR, Amplification of DelOP 72°C 2-step after cutting; run at 135 V, 0.8 % gel (TAE)(11.08)
Reaction
Reagent DelAF
Template D.acidovorans SPH-1 colony
Primer fw 4.5 µL FS_02
Primer rev 4.5 µL FS_05
DMSO 1 µL
Phusion Ready Mix 10 µL
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
67.5 - 65.0 (ΔT = 0.5) ↓ 0.5 5
72 3:20
18 98 1
65.5 - 63.0 (ΔT = 0.5) 5
72 3:20
1 72 10min
1 10 inf

Results:

  • Amplification of DelAF worked well, gradient displays an optimal annealing temperature of 65.5°C, which will be used for further amplifications
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit

13-08-2013

Restriction digest of fragment FS_02 to FS_05; 11.2 kb; 12-08-2013 with EcoRI-HF

Incubation at 37°C for 2 hours

what µL
FS_02 to FS_05 (12-08-2013) 20
EcoRI-HF 1
CutSmart Buffer 2.5
dd H2O 1.5

Expected fragment sizes: 2.26kbp; 4.62kbp; 4.32kbp

Results:

  • Weak bands of about 4.6kbp visible, as well as on of about 2.6kbp
  • digest will be repeated using higher concentrations of DNA to clearify results of restriction digest

Amplification from FS_02 to FS_05; 11.2 kb

Amplification of DelAF (FS_02-FS_05); run at 100 V, 0.8 % gel (TAE)(13.08)
Amplification of DelAF (FS_02-FS_05), cut; run at 100 V, 0.8 % gel (TAE)(13.08)
Reaction

1 sample contains 2µL of DMSO

Reagent DelAF
Template D.acidovorans SPH-1 colony
Primer fw 4.5 µL FS_02
Primer rev 4.5 µL FS_05
DMSO 1 µL
Phusion Ready Mix 10 µL
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
65.5 ↓ 0.5 5
72 3:20
18 98 1
63.5 5
72 3:20
1 72 10min
1 10 inf

Results:

  • Amplification of DelAF worked though a slight smear occured, therefore PCR will be repeated on the more precise Biometra TProfessional Basic cylcer again
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit

14-08-2013

Concentration measurement (FS_02 to FS_05; 11.2 kb)

Fragment Primer Date PCR Concentration
DelAF FS02-FS05 11-08-2013 ~10 ng/µL
DelAF FS02-FS05 12-08-2013 0 ng/µL
DelAF FS02-FS05 12-08-2013 0 ng/µL

Results:

  • concentrations are not sufficient for gibson assembly
  • PCR will be repeated and gel slices of different reactions will be pooled for one gel extraction using QIAquick Gel Extraction Kit to obtain the concentrations needed for gibson assembly

Restriction digest of fragment FS_02 to FS_05; 11.2 kb; 11-08-2013 with EcoRI-HF

Test restriction digest of fragment FS_02-FS_05 (12-08) with EcoRI; run at 100 V, 0.8 % gel (TAE)(13.08)


Incubation at 37°C for 2 hours 45min

what µL
FS_02 to FS_05 (11-08-2013) 18
EcoRI-HF 1
CutSmart Buffer 2.5
dd H2O 3.5

Expected fragment sizes: 2.26kbp; 4.62kbp; 4.32kbp

Results:

  • One band of about 5kbp and one of 2.5kbp
  • no clear result, digest will be repeated with another enzyme (ClaI), as the enzyme used was beyond expiration date

Restriction digest of fragment FS_02 to FS_05; 11.2 kb; 10-08-2013 with ClaI

Test restriction digest of fragment FS_02-FS_05 (12-08) with ClaI; run at 100 V, 0.8 % gel (TAE)(14.08)

Incubation at 37°C for 2 hours

what µL
FS_02 to FS_05 (10-08-2013) 20
ClaI 1
CutSmart Buffer 2.5
dd H2O 1.5

Expected fragment sizes: 6.9kbp, 4.3kbp

Results:

  • restriction digest displays the expected fragments, therefore amplification of the desired fragment can be assumed
  • PCR product will be prepared for sequencing by GATC to proof amplification of the desired DNA sequence before Gibson Assembly

17-08-2013

Amplification from FS_02 to FS_05; 11.2 kb

Amplification of DelAF (17-08); run at 100 V, 0.8 % gel (TAE)
Amplification of DelAF, cut (17-08); run at 100 V, 0.8 % gel (TAE)
Reaction of DelAF

6x20 µL

Reagent DelAF
Template D.acidovorans SPH-1 colony
Primer fw 2.5 µL FS_02
Primer rev 2.5 µL FS_05
Phusion Ready Mix 10 µL
DMSO 1 µL
dd H2O 4 µL
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
65.5 ↓ 0.5 5
72 3:20
18 98 1
63.5 5
72 3:20
1 72 10min
1 10 inf

Results:

  • Amplification of DelAF worked
  • bands were cut out, pooled and DNA purified using QIAquick Gel Extraction Kit to obtain concentrations needed for gibson assembly

18-08-2013

Amplification from FS_02 to FS_05; 11.2 kb

Amplification of DelAF (18-08); run at 100 V, 0.8 % gel (TAE)
Amplification of DelAF, cut (18-08); run at 100 V, 0.8 % gel (TAE)
Reaction of DelAF

6x20 µL

Reagent DelAF
Template D.acidovorans SPH-1 colony
Primer fw 2.5 µL FS_02
Primer rev 2.5 µL FS_05
DMSO 1 µL
Phusion Ready Mix 10 µL
dd H2O 4 µL
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
65.5 ↓ 0.5 5
72 3:20
18 98 1
63.5 5
72 3:20
1 72 10min
1 10 inf

Results:

  • Amplification of DelAF worked
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit

12-08-2013

Amplification from FS_02 to FS_11; 22.8 kb

Amplification of DelAG (12-08-13); run at 100 V, 0.8 % gel (TAE)
Reaction
Reagent DelAG
Template D.acidovorans SPH-1 colony
Primer fw 4.5 µl FS_02
Primer rev 4.5 µl FS_11
DMSO 1 µl
Phusion Ready Mix 10 µl


Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
72.0 - 67.0 (ΔT = 0.5) ↓ 0.5 5
72 7:20
18 98 1
70.0 - 65.0 (ΔT = 0.5) 5
72 7:20
1 72 10min
1 10 inf

Results:

  • none of the amplifications led to yields of DNA sufficient for Gibson Assembly, therefore the used combination of primers seems not to be sufficient for amplification of 22.0 kbp

13-08-2013

Amplification from FS_02 to FS_11_short; 22.8 kb

Reaction
Reagent DelAG
Template D.acidovorans SPH-1 colony
Primer fw 4.0 µl FS_02
Primer rev 4.0 µl FS_11_short
DMSO 2 µl
Phusion Ready Mix 10 µl


Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
72.0 - 67.0 (ΔT = 0.5) ↓ 0.5 5
72 7:20
18 98 1
70.0 - 65.0 (ΔT = 0.5) 5
72 7:20
1 72 10min
1 10 inf

15-08-2013

Amplification from SR_01 to FS_23; 6.2 kb

Amplification of DelG; run at 100 V, 0.8 % gel (TAE)(14.08)
Amplification of DelG; run at 100 V, 0.8 % gel (TAE)(14.08)
Reaction (5x20 µl)
what µl
D. acidovorans SPH-1 colony 1
SR_01: (1/10) 2
FS_23: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
66 ↓ 0.5 5
72 3:20
18 98 1
64 5
72 3:20
1 72 10min
1 12 inf

Results:

  • Amplification of DelG was successful
  • Bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • Amplification will be repeated to increase the concentration for the Gibson Assembly

16-08-2013

Concentration measurement

the fragment was gel purified

Fragment Primer Date PCR Concentration
DelG SR01-FS23 15-08-2013 52.6 ng/µl
DelG after nucleotide removal SR01-FS23 15-08-2013 30 ng/µl

Amplification from SR_01 to FS_23; 6.2 kb

Amplification of DelG ; run at 100 V, 0.8 % gel (TAE)(14.08)
Amplification of DelG ; run at 100 V, 0.8 % gel (TAE)(14.08)
Reaction (5x20 µl)
what µl
D. acidovorans SPH-1 colony 1
SR_01: (1/10) 2
FS_23: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
66 ↓ 0.5 5
72 3:20
18 98 1
64 5
72 3:20
1 72 10min
1 12 inf

Results:

  • Amplification of DelG was successful
  • Bands were cut out and DNA purified using QIAquick Gel Extraction Kit

12-08-2013

Amplification I from FS_22 to FS_13; 2.7 kb

Reaction
Reagent DelOP
Template D.acidovorans SPH-1 colony 1µl FS_22-FS_13_short 10-08
Primer fw 2 µl FS_22
Primer rev 2 µl FS_13
Phusion Ready Mix 10 µl
dd H2O 5 µl
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
55 5
72 1:00
1 72 5min
1 10 inf

Results:

  • Amplification of DelOP did not work
  • PCR will be repeated at very high annealing temperatures to ensure that primers with very complex secondary structure such as the Gibson-Primer FS_13long are able to bind

Amplification II and III from FS_22 to FS_13; 2.7 kb

Amplification of DelOP III .; run at 100 V, 0.8 % gel (TAE)(12.08)


Amplification of DelOP 72°C const.; run at 135 V, 0.8 % gel (TAE)(11.08)
Amplification of DelOP 72°C const. after cutting; run at 135 V, 0.8 % gel (TAE)(11.08)
Reaction
Reagent DelOP
Template 1µl FS_22-FS_13_short 10.08
Primer fw 4 µl FS_22
Primer rev 4 µl FS_13
Phusion Ready Mix 10 µl
DMSO 1 µl
Conditions
Biorad MyCycler
Cycles temperature [°C] DelOP II Time Cycles temperature [°C] DelOP III Time
1 98 10 s 1 98 10 s
30 98 1 s 12 98 1 s
74.0 - 70.0 (ΔT = 2.0) ↓ 0.5 5 s
72 1:00 min 72 3:15 min
18 98 1 s
66 5 s
68 / 70 / 72 1:00 min
1 72 10 min 1 72 10 min
1 10 inf 1 10 inf

Results:

  • Amplification of DelOP was successful, 2-step PCR with FS_22-FS_13s as template led to expected bands as well as a slight smear and an unintended product
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • PCR will be repeated to obtain more sample

Re-PCR from FS_22 to FS_13; 2.7 kb; 10-08-2013)

Amplification of DelAF using gradient PCR, Amplification of DelOP 72°C 2-step; run at 135 V, 0.8 % gel (TAE)(11.08)
Amplification of DelAF using gradient PCR, Amplification of DelOP 72°C 2-step after cutting; run at 135 V, 0.8 % gel (TAE)(11.08)


3x 20µl

Reaction
Reagent DelOP
Template 1µl FS_22-FS_13_short 10-08-2013
Primer fw 4 µl FS_22
Primer rev 4 µl FS_13
Phusion Ready Mix 10 µl
DMSO 1 µl
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72 1:05
1 72 5 min
1 10 inf

Results:

  • Amplification of DelOP was successfull
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • gradient PCR with annealing at 74°C, 72°C and 70°C will be carried out to increase yield

Amplification V from FS_22 to FS_13; 2.7 kb

Amplification of DelOP V (12-08-13); run at 100 V, 0.8 % gel (TAE)
Amplification of DelOP V (12-08-13) cut
Reaction
Reagent DelOP
Template D.acidovorans SPH-1 colony
Primer fw 4.5 µl FS_22
Primer rev 4.5 µl FS_13
Phusion Ready Mix 10 µl
DMSO 1 µl
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
75.0 - 72.0 (ΔT = 0.5) 1:05
1 72 5 min
1 10 inf

Results:

  • Amplification of DelOP was successfull
  • The gradient PCR shows that amplification worked best with an annealing temperature of 72.3°C, interestingly this annealing temperature was used for the remaining mastermix of the gradient PCR which included more DMSO than the other samples as mixing of DMSO often is not perfect and therefore the very last sample is of higher DMSO concentration
  • Therefore PCR will be repeated with an annealing temperature of 72.3°C and 10% DMSO

13-08-2013

Restriction digest of fragment FS_22 to FS_13; 2.7 kb; 12-08-2013) with EcoRI-HF

Incubation at 37°C for 2 hours

what µl
FS_22 to FS_13 (12-08-2013) 20
EcoRI-HF 1
CutSmart Buffer 2.5
dd H2O 1.5
Expected fragment lengths [bp] 1883, 960


Results:

  • restriction digest shows the expected bands, therefore validation if PCR amplicon is sufficient for Gibson Assembly

Amplification from FS_22 to FS_13; 2.7 kb

Amplification of DelAF (FS_02-FS_05); run at 100 V, 0.8 % gel (TAE)(13.08)
Amplification of DelAF (FS_02-FS_05), cut; run at 100 V, 0.8 % gel (TAE)(13.08)
Reaction
Reagent DelOP
Template D.acidovorans SPH-1 colony
Primer fw 4 µl FS_22
Primer rev 4 µl FS_13
Phusion Ready Mix 10 µl
DMSO 1/2 µl
dd H2O 1/- µl
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72 1:05
1 72 5 min
1 10 inf

Results:

  • Amplification of DelOP was successfull with 10% DMSO leading to one specific band of intended size
  • PCR will be repeated with 10% DMSO to obtain the amount of product which is necessary for Gibson Assembly and test restriction digest

14-08-2013

Concentration measurement DelOP

Fragment Primer Date PCR Concentration
DelOP FS22-FS13 12-08-2013 11 ng/µl

17-08-2013

Amplification from FS_22 to FS_13; 2.7 kb

6x20 µl

Reaction
Reagent DelOP
Template D.acidovorans SPH-1 colony
Primer fw 2.5 µl FS_22
Primer rev 2.5 µl FS_13
Phusion Ready Mix 10 µl
DMSO 2 µl
dd H2O 3 µl


Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72.3 5
72 1:00
1 72 5 min
1 10 inf

18-08-2013

Amplification from FS_22 to FS_13; 2.7 kb

Restriction digest of pSB4K5 (17-08) with DpnI (17-08) and amplification of DelOP (18-08); run at 100 V, 0.8 % gel (TAE)
Restriction digest of pSB4K5 (17-08) with DpnI (17-08) and amplification of DelOP, cut (18-08); run at 100 V, 0.8 % gel (TAE)

6x20 µl

Reaction
Reagent DelOP
Template D.acidovorans SPH-1 colony
Primer fw 2.5 µl FS_22
Primer rev 2.5 µl FS_13
Phusion Ready Mix 10 µl
DMSO 2 µl
dd H2O 3 µl


Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72.3 5
72 1:00
1 72 5 min
1 10 inf

Results:

  • Amplification of DelOP was successful
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit

15-08-2013

Amplification from FS_01 to FS_16; 4.2 kb

Amplification of DelFG and Backbone pSB4K5; run at 100 V, 0.8 % gel (TAE)(14.08)
Amplification of DelFG and Backbone pSB4K5; run at 100 V, 0.8 % gel (TAE)(14.08)
Reaction

(2x50µl)

what µl
Template pSB4K5 1
FS_01: (1/10) 2.5
FS_16: (1/10) 2.5
Phusion Master Mix 25
DMSO 2.5
dd H2O 16.5


Conditions
Biorad T100
Cycles-PCR temperature [°C] Time [s]
1 98 5
12 98 1
62 5
72 1:30 min
1 72 5 min
1 8 inf

Results:

  • Amplification worked very well, we had bright bands on the right height.
  • Bands were cut out and DNA purified using QIAquick Gel Extration Kit.

Restriction digest of pSB4K5 (FS_01 to FS_16; 4.2 kb; 15-08-2013) with DpnI

Incubation at 37°C for about 6 hours

what µl
FS_16 to FS_01 (15-08-2013) 30
DpnI 2
CutSmart Buffer 4
dd H2O 4

Results:

  • Bands were cut out and DNA purified using QIAquick Gel Extration Kit.

16-08-2013

Concentration measurement

The concentration of the gel purified, and DpnI digested fragment was measured using a NanoDrop Instrument.

Fragment Primer Date PCR Concentration
pSB4K5 DpnI digested FS01-FS16 15-08-2013 30 ng/µl

Amplification from FS_01 to FS_16; 4.2 kb

Amplification of pSB4K5 (16-08); run at 100 V, 0.8 % gel (TAE)
Amplification of pSB4K5; cut (16-08); run at 100 V, 0.8 % gel (TAE)
Reaction

(3x50µl)

what µl
Template pSB4K5 1
FS_01: (1/10) 2.5
FS_16: (1/10) 2.5
Phusion Master Mix 25
DMSO 2.5
dd H2O 16.5


Conditions
Biorad T100
Cycles-PCR temperature [°C] Time [s]
1 98 5
12 98 1
62 5
72 1:30 min
1 72 5 min
1 8 inf


Results:

  • Amplification worked very well, we had bright bands on the right height.
  • Bands were cut out and DNA purified using QIAquick Gel Extration Kit.

17-08-2013

Amplification from FS_01 to FS_16; 4.2 kb

Reaction

(3x50µl)

what µl
Template pSB4K5 1
FS_01: (1/10) 2.5
FS_16: (1/10) 2.5
Phusion Master Mix 25
DMSO 2.5
dd H2O 16.5


Conditions
Biorad MyCycler
Cycles-PCR temperature [°C] Time [s]
1 98 5
12 98 1
62 5
72 1:30 min
1 72 5 min
1 8 inf


Results:

  • Amplification worked very well, we had bright bands on the right height.
  • Bands were cut out and DNA purified using QIAquick Gel Extration Kit.

Restriction digest of pSB4K5 (FS_01 to FS_16; 4.2 kb; 17-08-2013) with DpnI

Restriction digest of pSB4K5 (17-08) with DpnI (17-08) and amplification of DelOP (18-08); run at 100 V, 0.8 % gel (TAE)
Restriction digest of pSB4K5 (17-08) with DpnI (17-08) and amplification of DelOP, cut (18-08); run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for about 6 hours

what µl
FS_16 to FS_01 (17-08-2013) 30
DpnI 2
CutSmart Buffer 4
dd H2O 4

Results:

  • Bands were cut out and DNA purified using QIAquick Gel Extration Kit.

18-08-2013

Amplification from FS_01 to FS_16; 4.2 kb

Reaction

(3x50µl)

what µl
Template pSB4K5 1
FS_01: (1/10) 2.5
FS_16: (1/10) 2.5
Phusion Master Mix 25
DMSO 2.5
dd H2O 16.5


Conditions
Biorad MyCycler*
Cycles-PCR temperature [°C] Time [s]
1 98 5
12 98 1
62 5
72 1:30 min
1 72 5 min
1 8 inf

Restriction digest of pSB4K5 (FS_01 to FS_16; 4.2 kb; 18-08-2013) with DpnI

Incubation at 37°C for about 6 hours

what µl
FS_16 to FS_01 (18-08-2013) 30
DpnI 2
CutSmart Buffer 4
dd H2O 4

Results:

  • Digested fragment was run on a gel.
  • Bands were cut out and DNA purified using QIAquick Gel Extration Kit.

13-08-2013

Amplification from FS_21 to FS_07; 5.2 kb

Amplifcation of DelFG (13-08); run at 100 V, 0.8 % gel (TAE)
Reaction
Reagent µl
Template D.acidovorans SPH-1 colony
Primer fw 4 µl FS_21
Primer rev 4 µl FS_07
Phusion Ready Mix 10 µl
DMSO 1 µl
dd H2O 1 µl
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
55 2:00
1 72 10 min
1 10 inf

Results:

  • Amplification of DelFG worked, but amount of DNA was insufficient
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • PCR will be carried out with another cylcer which by experience of the last amplifications led to higher product yields.

15-08-2013

Amplification from FS_21 to FS_26 ; 5.5 kb

Amplification of DelFG and Backbone pSB4K5; run at 100 V, 0.8 % gel (TAE)(14.08)
Amplification of DelFG and Backbone pSB4K5; run at 100 V, 0.8 % gel (TAE)(14.08)
Reaction

(5x20 µl)

what µl
D. acidovorans SPH-1 colony 1
FS_21: (1/10) 2
FS_26: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
60 5
72 2:15
1 72 10 min
1 10 inf

Results:

  • Amplification of DelFG was successful
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • obtained DNA will consequently be used for Gibson Assembly
  • PCR will be repeated to get higher amounts of DNA and increase concentration by purifying several gel slices using the same QIAquick spin column

16-08-2013

Concentration measurement DelFG

all fragments were gel purified, FG were additionally purified with the nucleotide removal kit

Fragment Primer Date PCR Concentration
DelFG FS21-FS26 29-07-2013 61.4 ng/µl
DelFG after nucleotide removal FS21-FS26 29-07-2013 30 ng/µl

Amplification from FS_21 to FS_26 ; 5.5 kb

Amplification of DelFG ; run at 100 V, 0.8 % gel (TAE)(14.08)
Amplification of DelFG ; run at 100 V, 0.8 % gel (TAE)(14.08)
Reaction

(5x20 µl)

what µl
D. acidovorans SPH-1 colony 1
FS_21: (1/10) 2
FS_26: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
60 5
72 2:15
1 72 10 min
1 10 inf

Results:

  • Amplification of DelFG was successful
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • obtained DNA will consequently be used for Gibson Assembly

15-08-2013

Gibson Assembly and electroporation

Assembly of our fragments

Those gel-purified fragments were added for the Gibson assembly of our construct pFSN.

Fragment Primer Date Concentration (ng/µl) Volume (µl)
pSB4K5 FS_01 - FS_16 14-08-13 13 1.35
AF FS_02 - FS_05 13-08-13 49.75 2.65
FG FS_21 - FS_26 07-08-13 24.5 2.69
G FS_35_SR_01 - FS_23 08-08-13 34.9 2.1
OP FS_22 - FS_13 13-08-13 36.3 0.89
L FS_14 - FS_15 07-08-13 51 0.31
Gibson MasterMix NEB 2x 10
  • Gibson Assembly according to protocols of NEB, 1h at 50°C

Gibson backbone (pSB4K5)

The backbone pSB4K5 was also incubated with the Gibson mastermix. We will use this as one negative control in the electroporation to test the efficiency.

Fragment Date Concentration (ng/µl) Volume (µl)
pSB4K5 14-08-13 13 1.35
H2O 8.65
Gibson MasterMix NEB 2x 10
  • Gibson Assembly according to protocols of NEB, 1h at 50°C

Electroporation

The following samples were electroporated

mixture volume used cells
5 µl Gibson Assembly of pFSN construct 10 µl H2O 1 µl 50 µl DH10β old
5 µl Gibson Assembly of pFSN construct 10 µl H2O 14 µl 50 µl DH10β old
5 µl Gibson Assembly of Backbone (control) 10 µl H2O 1 µl 50 µl DH10β old
pSB4K5 14-08-13 1.35 µl 50 µl DH10β old
pSB6A1 Hanna [69.57 ng/µl] 1:10 1 µl 50 µl DH10β Fanny new


  • Cells were transdered into 400 µl SOC-medium (NEB) after electroporation
  • incubation and growth of cells for 1 hour at 37°C
  • cells were centrifuged for 3 min at 6000rpm, supernatant discarded and cells resuspended in remaining SOC-medium
  • cells were plated on agar plates containing kanamycine, 10µl on one plate, remaining µl on another plate for electroporation of the construct pFSN and on agar plates containing ampicillin for electroporation of the control backbone pSB6A1


20130816ElectroporationA.JPG

1 µl of diluted Gibson electroporated; 10 µl of E.coli plated

20130816ElectroporationB.jpg

1 µl of diluted Gibson electroporated; Rest of E.coli plated

20130816ElectroporationC.jpg

14 µl of diluted Gibson electroporated; 10 µl of E.coli plated

20130816ElectroporationD.jpg

14 µl of diluted Gibson electroporated; Rest of E.coli plated

P1010689.JPG

1 µl of diluted Gibson (backbone control) electroporated; 10 µl of E.coli plated

P1010690.JPG

1 µl of diluted Gibson (backbone control) electroporated; Rest of E.coli plated

P1010692.JPG

1 µl of backbone control electroporated; 10 µl of E.coli plated

P1010693.JPG

1 µl of backbone control electroporated; Rest of E.coli plated

P1010704.JPG

Test of electrocompetence DH10β with 1 µl pSB6A1; 10 µl of E.coli plated

P1010705.JPG

Test of electrocompetence DH10β with 1 µl pSB6A1; Rest of E.coli plated

16-08-2013

Colony PCRs

As a lot of colonies grew we screened if DelL and pSB4K5 are present.

File:20130816 2log screeningsophieDelRESTbesch.png
Colony PCRs of red colonies after gibson assembly and electroporation (16-08); run at 100 V, 0.8 % gel (TAE)

Different probes:

  • A: 1 µl of diluted Gibson electroporated; 10 µl of E.coli plated
  • B: 1 µl of diluted Gibson electroporated; Rest of E.coli plated
  • C: 14 µl of diluted Gibson electroporated; 10 µl of E.coli plated
  • D: 14 µl of diluted Gibson electroporated; Rest of E.coli plated


  • Only red colonies were chosen for screening
  • The expected amplicon size is 1.5 kb


Reaction mixture
Reagent A1, A2 B1-B5 C1-C3 D1-D40
VR-Primer: (1/10) 2 µl 2 µl 2 µl 2 µl
FS_14: (1/10) 2 µl 2 µl 2 µl 2 µl
Colonies 2 red colonies A (A1, A2) 5 red colonies B (B1-B5) 3 red colonies C (C1-C3) 40 red colonies D (D1-D40)
DreamTaq 10 µl 10 µl 10 µl 10 µl
dd H2O 5 µl 5 µl 5 µl 5 µl


PCR Conditions

--> T100

Cycles-PCR temperature [°C] Time [s]
1 95 2:00
12 95 1:00
66 ↓ 0.5 5
72 1:30 min
18 95 1:00
63 5
72 1:30 min
1 12 inf

Result:

  • None of the screened colonies led to the correct amplicon, therefore the correct assembly has not worked in any of the white colonies
  • screening will be repeated, with white colonies as template, as bacteria including the correct 32 kbp backbone might not be capable of expressing mRFP anymore due to the large insert between lac promotor and mRFP

17-08-2013

Colony PCRs

File:20130817 2log DelRestwhiteColonyPCRsbesch.png
Colony PCRs of white colonies after gibson assembly and electroporation (17-08); run at 100 V, 0.8 % gel (TAE)

Different probes:

  • A 1 µl of diluted Gibson Assembly electroporated; 10 µl of E.coli plated
  • B 1 µl of diluted Gibson Assembly electroporated; remaining E.coli plated
  • C 14 µl of diluted Gibson Assembly electroporated; 10 µl of E.coli plated
  • D 14 µl of diluted Gibson Assembly electroporated; remaining E.coli plated


  • Only white colonies where chosen for screening
  • The expected amplicon size is 1.5 kb


Reaction mixture
Reagent B1w C1w D1w-D10w
VR-Primer: (1/10) 2 µl 2 µl 2 µl
FS_14: (1/10) 2 µl 2 µl 2 µl
Colonies 1 white colony B (B1w) 1 white colony C (C1w) 10 white colonies D (D1w-D10w)
DreamTaq 10 µl 10 µl 10 µl
dd H2O 5 µl 5 µl 5 µl


PCR Conditions

--> T100

Cycles-PCR temperature [°C] Time [s]
1 95 2:00
12 95 1:00
66 ↓ 0.5 5
72 1:30 min
18 95 1:00
63 5
72 1:30 min
1 12 inf

Result:

  • Colony D8w shows expected amplicon of 1.5kbp
  • The other colonies were screened negative.
  • D8w will be transfered to liquid culture to repeat screening PCR as well as for further validation beeing screening for the other insert fragments as well as test restriction digest
  • Furthermore another electorporation will be carried out to yield more clones positive for screening with the Primers VR and FS_14, therefore the remaining 10µl of the Gibson assembly will be purified using isopropanol precipitation


Electroporation

We electroporated the Gibson assembly (15-08) again to increase the chance of a positive clone. We purified it by isopropanol purification.

mixture volume used Cells
Assembled fragments (15-08) isopropanol precipitated 15 µl 50 µl DH10β


  • Cells were transdered into 400 µl SOC-medium (NEB) after electroporation
  • incubation and growth of cells for 1 hour at 37°C
  • cells were centrifuged for 3 min at 6000rpm, supernatant discarded and cells resuspended in remaining SOC-medium
  • cells were plated on agar plates containing kanamycine, 10µl on one plate, remaining µl on another plate for electroporation of the construct pFSN
P1010701.JPG

15 µl of Gibson 15-08 (isopropanol precipitated) electroporated; 10 µl of E.coli plated

P1010702.JPG

15 µl of Gibson 15-08 (isopropanol precipitated) electroporated; Rest of E.coli plated

18-08-2013

Colony PCRs

File:20130818 log2 D8w D11w-D23w E1w-E19w E1r-E10r F1w-F28w F1r-F133 F19r F16r.png
Colony PCRs to test if pSB4K5 and DelL are ligated; run at 100 V, 0.8 % gel (TAE)(14.08)

Different probes:

  • D: 14 µl of diluted Gibson Assembly electroporated; remaining E.coli plated
  • E: 1 µl isopropanol precipitated Gibson Assembly (15-08) ; 10 µl of E.coli plated
  • F: 1 µl isopropanol precipitated Gibson Assembly (15-08); remaining E.coli plated
  • The expected amplicon size is 1.5 kb
  • 3 colonies were sceened in one PCR
Reaction mixture
Reagent D8w D11w-D24w E1w-E19w F1w-F20w E1r-E10 F1r-F20r
VR-Primer: (1/10) 2 µl 2 µl 2 µl 2 µl 2 µl 2 µl
FS_14: (1/10) 2 µl 2 µl 2 µl 2 µl 2 µl 2 µl
Colonies Colony D8w liquid culture 24 white colonies D (D1w-D24w) 19 white colonies E (E1w-E19w) 30 white colonies F (F1w-F20w) 10 red colonies E (E1r-E10r) 20 red colonies F (F1r-F20r)
DreamTaq 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl
dd H2O 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl


PCR Conditions

--> T100

Cycles-PCR temperature [°C] Time [s]
1 95 2:00
12 95 1:00
66 ↓ 0.5 5
72 1:30 min
18 95 1:00
63 5
72 1:30 min
1 12 inf

Result:

  • colony D8w again showed the expected amplicon in the colony PCR and will therefore be further analyzed for the other fragments as well as restriction digested and partially sequenced as soon as screening primers arive
  • Furthermore colonies E16w and F25w are screened positive for the fragment DelL

19-08-2013

Concentration measurement

The concentration of the gel purified, and DpnI digested fragments was measured using a NanoDrop Instrument.

Fragment Primer Date PCR Concentration
pSB4K5 DpnI digested FS_01-FS_16 17-08-2013 101.2 ng/µl
pSB4K5 DpnI digested FS_01-FS_16 18-08-2013 159.2 ng/µl

19-08-2013

Colony PCRs for testing ligation of pSB4K5 and DelL

Reaction mixture
Reagent E13w E14w E15w F25w F26w F27w
VR-Primer: (1/10) 2 µl 2 µl 2 µl 2 µl 2 µl 2 µl
FS_14: (1/10) 2 µl 2 µl 2 µl 2 µl 2 µl 2 µl
Colonies Colony E13w liquid culture Colony E14w liquid culture Colony E15w liquid culture Colony F25w liquid culture Colony F26w liquid culture Colony F27w liquid culture
DreamTaq 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl
dd H2O 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl

Result:

  • Colony F26w was screened positive, as it shows the expected band at 1.4kbp, the others were screened negative
  • 8 ml of colony D8w and colony D26w (pFSN-1) were midi-preped according to protocol 'Preparation of large plasmids'.


Colony PCRs for testing ligation of pSB4K5 and DelAF, as well as DelAF and DelFG

Sceening of colonies D8w and D26w for fragments pSB4K5, DelAF, and DelFG.
Reaction mixture
Reagent D8w D8w F26w F26w
Primer_fw (1/10) 2 µl FS_47 2 µl FS_51 2 µl FS_47 2 µl FS_51
Primer_rv (1/10) 2 µl FS_48 2 µl FS_52 2 µl FS_48 2 µl FS_52
Colonies Colony D8w liquid culture Colony D8w liquid culture Colony F26w liquid culture Colony F26w liquid culture
DreamTaq 10 µl 10 µl 10 µl 10 µl
dd H2O 5 µl 5 µl 5 µl 5 µl
Expected band 547 bp 724 bp 547 bp 724 bp

Results:

  • The screening was positive for clone D8w with both primer combinations.
  • Thus this clone contains the backbone pSB4K5, the genes DelA, DelB, DelC, DelD, DelE, DelF, DelL and at least a part of the gene DelG.
  • For colony F26w the screenings were negativ.

Restriction digest of pFSN (31.435 kbp)

Restriction digest of pFSN (19-08) with l2:EcoRI, l3:BamHI, l4:BglII (17-08); run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for about 6 hours

Reagent µl
pFSN 1
EcoRI BamH1 BglII 2
CutSmart Buffer 0.5
dd H2O 1.5
Expected fragment lengths [bp] EcoRI 9798, 7856, 5276, 4624, 2529, 1212
Expected fragment lengths [bp] BamHI 23860, 7435
Expected fragment lengths [bp] BglII 14223, 11919, 3291, 1892

Results:

  • All digests displayed the expected bands and thus were positive.
  • Therefore the pFSN plasmid is very likely our desired construct.

20-08-2013

Sequencing

  • The construct pFSN was isolated from overnight culture using the 'Preparation of large plasmids' protocol.
  • Single read sequencing was carried out by GATC Biotech
  • obtained .abi files were analyzed with UniPro Ugene, part of sequence showing convenient chromatogram was chosen for alignment using ClustalOmega


Sequence-Files from GATC Biotech

Sequencing of pFSN-1 with Primer FS_13_short

Sequencing of pFSN-1 with Primer FS_22

Sequencing of pFSN-1 with Primer VFII

Sequencing of pFSN-1 with Primer VR

Alignments

Alignment for sequencing of pFSN with primer FS_13s_rv against the expected construct to validate sequence of DelOP

Alignement for sequencing of pFSN with primer FS_57_rv against the expected construct to validate the sequence for the overlap of DelOP to DelG

Alignement for sequencing of pFSN with primer FS_58_fw against the expected construct to validate the sequence for the overlap of DelOP to DelL

Alignement for sequencing of pFSN with primer FS_63_rv against the expected construct to validate the sequence for the overlap of DelL to mRFP

Alignement for sequencing of pFSN with primer VF2 against the expected construct to validate the sequence for the overlap of pSB4K5 to DelAF


Results:

  • Sequencing of the final construct pFSN for all internal Gibson sites validated the intended sequence
  • Sequence of mRFP shows mutations within the primer site, therefore it can be concluded that mRFP is not functional
  • Colonies of D8w will be further analyzed by FACs to investigate whether mRFP is functional or not
  • Additionaly, SDS-page of clone D8w will be conducted to check for expression of the inserted Del genes
  • Concluding the results of colony PCRs, restriction digests and sequencing it can be concluded, that we successfully amplified 28 kbp of genomic DNA from our donator organism Delftia acidovorans SPH-1 distributed from the DSMZ, ligated the thereby obtained 6 fragments including the standard biobrick backbone pSB4K5 sucessfully in the intended order and consequently transformed a plasmid of 32 kbp in total into E. Coli using electroporation

21-08-2013

  • Another 8 ml of colony D8w (pFSN-1) were midi-preped according to protocol 'Preparation of large plasmids'.

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