Team:Heidelberg/Templates/DelH week19

From 2013.igem.org

Contents

02-09 - 08-09-13

Characterization of DelH Plasmid pHM04 30-08 Clone 12

Maxiprep

  • 1 ml of an ON culture DH10ß-DelH were inocculated in 200 ml LB Amp and cultured ON at 37°C.
  • Maxi prep was performed by upscaling of mini precipitation protocol.

Result

The maxi prep yielded 6x 180 µl.

Maxi prep Concentration [µg/µl]
1 5.40
2 5.37
3 5.42
4 5.40
5 5.38
6 5.42
  • Sequencing showed a mutation at the beginning of DelH
Next steps
=> Sequencing the other 3 positive colonies => hopefully one of them has no mutation at the beginning of DelH
=> Sequence D. acidovorans, maybe the organism SPH1 was not perfectly sequenced => amplify with FS35 & DN07


Characterization of DelH Plasmid pHM04 30-08 Clones 4, 6, 15

Sequencing

  • Miniprep performed
  • Send in for sequencing => no reliable result
  • Maxiprep performed => Clone 4: c= 5.51 µg/µl, Clone 6: c= 3.71µg/µl, Clone 15: c= 4.65 µg/µl
  • Send in for sequencing with DN07 again

Result

Sample Alignment File
Midi-col 4 File:Heidelberg Sequencing Result pHM04 DN07 colony 04.clustal.txt
Midi-col 6 sequencing insufficient
Maxi-col 15 sequencing insufficient


Electroporation

Because the sequenced DelH colony 12 has a mutation at the beginning of the coding DNA, we are sequencing the other colonies and parallel we transformed with the rest of the same Gibson Mix (15 µl) used for the electroporation before (colonies 4, 6, 12, 15). In advance, we purified it via isoprop purification protocol.

Sample Isoprop purified Gibson Mix Colonies grown?
A 10 µl yes
B 20 µl yes (a lot more than on sample A)


Colony-PCR conditions CP.W19.A

There were 30 colonies screened. 3 per tube and in total 10 colonies of sample A & 20 colonies of sample B

Template 3 PC of S-A 3 PC of S-A 4 PC of S-A 3 PC of S-B 3 PC of S-B 4 PC of S-B 3 PC of S-B 3 PC of S-B 4 PC of S-B
Expected length [bp] 663 663 663 663 663 663 663 663 663
Named 1-3 4-6 7-10 11-13 14-16 17-20 21-23 24-26 27-30
Primer fw 10 µM 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2
Primer rev 10 µM 2 µl DN07 2 µl DN07 2 µl DN07 2 µl DN07 2 µl DN07 2 µl DN07 2 µl DN07 2 µl DN07 2 µl DN07
iTaq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl
Cycles Temperature DelH [°C] Time [s]
1 95 120
12 95 60
68 (touchdown -0.5°C) 30
72 45
18 95 60
65 30
72 45
1 12 inf

Result

Expected band: 663 bp

Fig.19.1 colony PCR of Gibson assembled DelH-BB without mRFP (loaded 20 µL of PCR)
l1:2log, l2:colonies 1-3, l3:colonies 4-6, l4:colonies 7-10, l5:colonies 11-13, l6:colonies 14-16, l7:colonies 17-20, l8:colonies 21-23, l9:colonies 24-26, l10:colonies 27-30
l4, l5,l8,l10:show the specific band at 663 bp

Some of the colony mixes show positive band.

=> Screen again colonies 7-10, 11-13, 17-20, 27-30 in a single PCR tubes.


Colony-PCR conditions CP.W19.B

Template 7 8 9 10 11 12 13 17 18 19 20 27 28 29 30
Expected length [bp] 663 663 663 663 663 663 663 663 663 663 663 663 663 663 663
Named 7 8 9 10 11 12 13 17 18 19 20 27 28 29 30
Primer fw 10 µM 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2
Primer rev 10 µM 2 µl DN07 2 µl DN07 2 µl DN07 2 µl DN07 2 µl DN07 2 µl DN07 2 µl DN07 2 µl DN07 2 µl DN07 2 µl DN07 2 µl DN07 2 µl DN07 2 µl DN07 2 µl DN07 2 µl DN07
iTaq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl
Cycles Temperature DelH [°C] Time [s]
1 95 120
12 95 30
68 (touchdown -0.5°C) 30
72 45
18 95 30
65 30
72 45
1 12 inf

Result

Expected band: 663 bp

Fig.19.3 colony PCR of Gibson assembled DelH-BB without mRFP (loaded 20 µL of PCR)
l1:50 bp ladder, l2:colonies 18, l3:colonies 19, l4:colonies 20, l5:colonies 27, l6:colonies 28, l7:colonies 29, l8:colonies 30
l3 and l7 show the expected band at 663 bp = col 19 & 29
Fig.19.2 colony PCR of Gibson assembled DelH-BB without mRFP (loaded 20 µL of PCR)
l1:50 bp ladder, l2:colonies 7, l3:colonies 8, l4:colonies 9, l5:colonies 10, l6:colonies 11, l7:colonies 12, l8:colonies 13, l9:colonies 17
l2 and l8show the expected band at 663 bp = col 7 & 13

Colonies 7,13, 19 & 29 show a definit result

=> Add medium and perform a mediprep the next day for sending in for sequencing.


Midiprep

Performed with 300 µl P1, 600 µl P2 and after incubating for 180 450 µl S3. Protocol was performed with isoprop/ethanol purification.

Sample Concentration [ng/µl]
Midi-col 7 2628.5
Midi-col 13 2379.3
Midi-col 19 2846.6
Midi-col 29 2846.6


Test Restriction Digest

We performed a test digest with EcoRI-HF of the screened samples col 7, col 13, col 19, col 29. We expect a band of 17.9 Kb and 4.9 Kb.

Sample Concentration [ng/µl] Amount in restriction digest EcoRI-HF [µl] CutSmartBuffer [µl] ddH2O [µl] Total amount [µl]
Midi-col 7 2628.5 0.5 µl = 1314.25 ng 1 2 16.5 20
Midi-col 13 2379.3 0.5 µl = 1189.65 ng 1 2 16.5 20
Midi-col 19 2846.6 0.5 µl = 1423.3 ng 1 2 16.5 20
Midi-col 29 2846.6 0.5 µl = 1423.3 ng 1 2 16.5 20
  • Incubation time: 1 h at 37 °C

Result

Expected bands: 17.9 Kb and 4.9 Kb

Fig.19.4. Test digest of pHM04 from different colonies with EcoRI-HF (loaded 20 µL of PCR)
l1:1kb+ ladder,l2:colony 7,l3:colony 19,l4:colony 29,l5:colony 13,
l2-3:show the expected bands at ~5 Kb and ~18 Kb

Colonies 7 & 13 show a definit result

=> Add medium and perform a mediprep the next day for send in for sequencing.


Sequencing

The colonies 7 & 19 were sent in MWG for sequencing. There for we prepared 15 µl of the plasmid (midiprep) with a concentration of 50-100 ng/µl and added 2 µl DN07 primer (10µM)

Sample Concentration [ng/µl] Amount for sequencing ddH2O added up to 15 µl Final concentration [ng/µl]
Midi-col 7 2628.5 0.5 µl = 1314.25 ng 14.5 87.62
Midi-col 19 2846.6 0.5 µl = 1423.3 ng 14.5 94.89
Maxi-col 4 5500 2 µl of 1:10 dilution = 1100 ng 13 73.3

Result

Sample Alignment File
Midi-col 7 File:Heidelberg Sequencing Result pHM04 DN07 colony 7.clustal.txt
Midi-col 19 File:Heidelberg Sequencing Result pHM04 DN07 colony 19-2.clustal.txt
Maxi-col 4 File:Heidelberg Sequencing Result pHM04 DN07 colony 04.clustal.txt


Test Restriction Digest

We performed a test digest with EcoRI-HF and PvuI-HF of the screened samples col 4, col 7, col 19. We expect a band of 17.9 Kb and 4.9 Kb for the digest with EcoRI-HF and bands of 11.5 Kb, 8.5 Kb and 2.6 Kb.

Sample Concentration [ng/µl] Amount in restriction digest Enzyme CutSmartBuffer [µl] ddH2O [µl] Total amount [µl]
Midi-col 4 5500 0.25 µl = 1375 ng EcoRI-HF 1 µl 2 16.75 20
Midi-col 4 5500 0.25 µl = 1375 ng PvuI-HF 1 µl 2 16.75 20
Midi-col 7 2628.5 0.5 µl = 1314.25 ng PvuI-HF 1 µl 2 16.5 20
Midi-col 19 2846.6 0.5 µl = 1423.3 ng PvuI-HF 1 µl 2 16.5 20
  • Incubation time: 1:20 h at 37 °C

Result

Expected band: 17.9 Kb and 4.9 Kb (EcoRI-HF) and 11.5 Kb, 8.5 Kb and 2.6 Kb (PvuI)

Fig.19.5. Test digest of pHM04 from different colonies with EcoRI-HF and PvuI (loaded 20 µL of PCR)
l1:1kb+ ladder,l2:colony 4 EcoRI,l3:colony 4 PvuI,l4:colony 7 PvuI,l5:colony 19 PvuI,
l2-3:show the expected bands

Colony 7 is the only colony which is positiv for all restriction digests.

=> Electroporate plasmid together with DelRest plasmid, as well as the Methylmanolyl-CoA plasmid.


Sequencing of D. acidovorans

PCR Conditions DA.W19.A

For being sure that the mutation at the beginning of DelH is not some mistake in the sequenced genome we amplify this part with one primer binding the delG (FS35) and the screening primer binding at the beginning of DelH (DN07) and let it sequence with GATC.

Template 1 µl glycerol stock of D. acidovorans 1 µl glycerol stock of D. acidovorans
Expected length [Kb] 6.5 6.5
Named TD 2step
Primer fw 10 µM 2 µl FS35 2 µl FS35
Primer rev 10 µM 2 µl DN07 2 µl DN07
Phusion Flash (2x) 10 µl 10 µl
ddH2O 4 µl 4 µl
DMSO 1 µl 1 µl
Cycles Temperature A [°C] Time [s] Temperature B[°C] Time [s]
1 98 10 98 10
12 98 1 98 1
68 (touchdown -0.5°C) 5 - -
72 130 72 120
18 98 1 98 1
66 5 - -
72 130 72 120
1 72 10 min 72 10 min
1 12 inf 12 inf

Result

Expected band: 6.5 Kb

Fig.19.7 Amplified fragments of DelG-DelH-beginning (loaded 20 µL of PCR)
l1:2log, l3-4PCR of 2 step, l5-6 PCR of 68 td
l3-6:show expected band at 7 Kb = was cut
Fig.19.6 Amplified fragments of DelG-DelH-beginning (loaded 20 µL of PCR)
l1:2log, l3-4PCR of 2 step, l5-6 PCR of 68 td
l3-6:show expected band at 7 Kb

Gel shows expected band.

=> Fragments were cut but not extracted, because side bands were cut too.
=> Run again PCR with 72.3 °C annealing temperature.


PCR Conditions DA.W19.B

Template 1 µl glycerol stock of D. acidovorans
Expected length [Kb] 6.5
Named FS35-DN07
Primer fw 10 µM 2 µl FS35
Primer rev 10 µM 2 µl DN07
Phusion Flash (2x) 10 µl
ddH2O 4 µl
DMSO 1 µl
Cycles Temperature [°C] Time [s]
1 98 10
30 98 1
72.3 5
72 120
1 72 10 min
1 12 inf

Result

Expected band: 6.5 Kb

Fig.19.9 Amplified fragments of DelG-DelH-beginning (loaded 20 µL of PCR)
l1:1kb plus ladder, l2-4PCR with 72.3°C annealing :show expected band at 7 Kb = was cut
Fig.19.8 Amplified fragments of DelG-DelH-beginning (loaded 20 µL of PCR)
l1:1kb plus ladder, l2-4PCR with 72.3°C annealing :show expected band at 7 Kb

Gel shows expected band at ~7 Kb.

=> The fragment was cut and gel purified. Nevertheless the bands were rather weak, so the PCR was repeated.


PCR Conditions DA.W19.C

Template 1 µl glycerol stock of D. acidovorans
Expected length [Kb] 6.5
Named FS35-DN07
Primer fw 10 µM 2 µl FS35
Primer rev 10 µM 2 µl DN07
Phusion Flash (2x) 10 µl
ddH2O 4 µl
DMSO 1 µl
Cycles Temperature [°C] Time [s]
1 98 10
30 98 1
72.3 5
72 120
1 72 10 min
1 12 inf

Result

Expected band: 6.5 Kb

Fig.19.11 Amplified fragments of DelG-DelH-beginning (loaded 20 µL of PCR)
l1:1kb plus ladder, l2-4PCR with 72.3°C annealing :show expected band at 7 Kb = was cut out
Fig.19.10 Amplified fragments of DelG-DelH-beginning (loaded 20 µL of PCR)
l1:1kb plus ladder, l2-4PCR with 72.3°C annealing :show expected band at 7 Kb

Gel shows expected fragment.

=> The fragment was cut and gel purified.
=> Send in for sequencing with DN07.


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