Team:Heidelberg/Templates/DelH week22

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Contents

23-09 - 29-09-13

Mutagenesis of DelH Clone I 6B

Screening-PCR Conditions CP.W22.A

Screening of the 11 colonies, which grew on the agar plate.

Template 11x 1 picked colony + positve control (Midiprep I6B)
Expected length [bp] 319 (VFII+HM22); 395 (FS66+SR03)
Named Mut1-11 (VFII+HM22), Mut1-11 (FS66+SR03)
Primer fw 10 µM 2 µl VF2 / FS66
Primer rev 10 µM 2 µl HM22 / SR03
One-Taq (2x) 10 µl
ddH2O 6 µl
Cycles Temperature DelH [°C] Time [s]
1 95 900
35 95 30
55 30
72 25
1 12 inf

Result

Expected bands: 319 bp (HM22-VFII) and 395 bp (FS_66-SR_03)

Fig.22.1 Colony PCR for screening of DelH-pSB6A1 construct

10 ml of each colony were cultured.

  • The screening with primer FS_66 to SR_03 did not work. The positive control was negative.
  • The screening with primer VFII to HM22 was positive for 4 colonies (2,3,4,7,8,10)
  • These colonies have to be midi-preped and digested to test whether it is the original DelH or the mutated one


Midiprep of Clones 2,3,4,7,8 and 10

  • Centrifugation of 10 ml culures at 3750 rpm for 15 min
  • Kept pellet an resuspended in 400 µl P1 buffer
  • Added 800 µl P2 buffer and incubated for 180
  • Neutralized with S3 buffer
  • Centrifugated for 15 min at 13 000 rpm
  • Kept supernatant and added 50:50 Isopropanol


Test restriction digest of Midipreps with MfeI and XbaI

Expected band: 22.9 kb

Fig.22.2 Test restriction digest with XbaI and MfeI to see if mutagenesis has worked.
Reagent Volume [µl]
Midiprep colonies 0.2
Cutsmart buffer 2
XbaI 1.5
MfeI-HF 1.5
ddH2O 14.75
  • Incubation for 2 h at 37°C

Apparently the mutagenesis did not work. The band was too low. Maybe recombination occured.

Sequencing

The Midiprep of clone 2 was send to sequencing to see what had happened during the mutagenesis.

Sequencing results for colony 2 with primer VFII after mutagenesis.

Result

The mutagenesis did not work. Apparently recombination occured. As the result was the same for all the colonies we suspect that repeating the mutagenesis would lead to the same result. Therefore the mutagenesis part is stopped.

SDS-PAGE of Clone C5

Lysis of Cells

  • 1 ml over night cultures in LB of electrocompetent BL21-pLys and BL21-pLys with pHM04 (C5)
  • Meausured ODs (BL21-pLys = 1.33; BL21-pLys + pHM04= 1.03 )
  • Centrifugation at 13000 rpm for 10 min
  • Discarded supernatant
  • Resuspended cells in 133 µl (BL21-pLys) and 103 µl (BL21-pLys + pHM04) 1x loading buffer (40% ddH2O, 12.5% TrisHCl pH=7.5, 20% Glycerol (50%), 20% SDS (10%), 5% β-Mercaptoethanol, 2.5% Bromophenol blue)
  • Boiled samples for 10 min at 98°C

Electrophoresis

  • Applied 25 µl of each sample on SDS-polyacrylamide gel
  • Run at 100 V for 5 min and afterwards at 140 V for another 50 min

Staining and Destaining Procedure

  • Gel was stained in Coomassie Brilliant Blue for 1 h on shaker
  • Destaining with destaining buffer (50% ddH2O, 40% Methanol, 10% Acetic acid) until band were visible

Result

Fig.22.3: SDS page of DelH clone C5 in E. coli BL21 DE3

As shown in figure 22.3, there is a faint very high band, which is DelH (~600 kDa). Clone C5 expresses DelH despite of the amino acid substitution. Next, we will electroporate DelH C5 with DelRest and pIK8.6 in E. coli BL21 DE3 and analyze Delftibactin expression by Micro-TOF.

Generation of DelH Plasmid pHM04 18-09

Colony-PCR CP.W22.B

Template 48x 1 picked colony
Expected length [bp] 663
Named 1A - 12E, DelH III
Primer fw 10 µM 50x 2 µl VF2
Primer rev 10 µM 50x 2 µl DN07
One-Taq Polymerase (2x) 50x 10 µl
ddH2O 48x 6 µl
Cycles Temperature [°C] Time [s]
1 95 120
12 95 30
68 (touchdown -0.5°C) 30
72 45 s
18 95 30
65 30
72 45
1 12 inf

Result

Expected band: 663 bp

Fig.22.4 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II(loaded 4 µL of PCR)
l1:2-log ladder, l2-l26:colonies A1-B12
Fig.22.5 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II (loaded 4 µL of PCR)
l1:2-log ladder, l2-l26:colonies C1-D12
Fig.22.6 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II (loaded 4 µL of PCR)
l1:2-log ladder, l2-l26:colonies E1-F12
  • The following colonies seem positive, because they show the expected band at 663 bp
Picture Positive colonies
22.1 A9
22.2 D7, D9, D11
22.3 E5, E6, E7, F7

Test Restriction Digest

The positive screened colonies were digested with PvuI.

Template DNA CutSmart Buffer PvuI ddH2O Total Volume
8.5 µl 1 µl 0.5 µl - 10 µl

Result

Expected bands: 11,621, 8628 & 2,685 bp

Fig.22.7 Test restricion digest of colonies from plate II and III

The following colonies show the correct pattern:

Colonies Positive in Digest Concentration in Midiprep [ng/µl]
II A9 yes 2,153
III D7 yes 3,371
III D9 yes 697
III D11 yes 3,523
III E6 yes 2,745
III E7 yes 3,649

They were not sequenced due to exclusively found mutations in BL21 DE3 (pLys).

Generation of DelH Plasmid pHM04 20-09

Colony-PCR CP.W22.C

Template 48x 1 picked colony
Expected length [bp] 663
Named 1F - 12H, DelH III
Primer fw 10 µM 50x 2 µl VF2
Primer rev 10 µM 50x 2 µl DN07
One-Taq Polymerase (2x) 50x 10 µl
ddH2O 48x 6 µl
Cycles Temperature [°C] Time [s]
1 95 120
12 95 30
68 (touchdown -0.5°C) 30
72 45 s
18 95 30
65 30
72 45
1 12 inf

Result

Expected band: 663 bp

Fig.22.3 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II (loaded 4 µL of PCR)
l1:2-log ladder, l2-l26:colonies E1-F12
Fig.22.4 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II (loaded 4 µL of PCR)
l1:2-log ladder, l2-l26:colonies G1-H12
  • The following colonies seem positive, because they show the expected band at 663 bp
Picture Positive colonies
22.3 F7, F9, F11
22.4 G3, G4, G12, H6, H11

Test Restriction Digest

The positive screened colonies were digested with PvuI.

Template DNA CutSmart Buffer PvuI ddH2O Total Volume
8.5 µl 1 µl 0.5 µl - 10 µl
Result

Expected bands: 11,621, 8628 & 2,685 bp

Fig.22.x Test restriction digest of DelH colonies

The following colonies show the correct pattern:

Colonies Positive in Digest Concentration in Midiprep [ng/µl]
III F7 yes 3,856
III F9 yes 1,283
III F11 no 4,507
III G3 yes 5,823
III G4 yes 3,622
III G12 no 3,615
III H6 no 3,985
III H11 no 3,605


Sequencing

Colonies F7, F9, G3 and G4 were send for sequencing with VF2.

Result

Colonies Alignment File
III F7 Sequencing of clone III F7 with VF2
III F9 Sequencing of clone III F9 with VF2
III G3 Sequencing of clone III G3 with VF2
III G4 Sequencing of clone III G4 with VF2



Contamination of Cells with Chloramphenicol Resistance

Preparation of Electrocompetent BL21 DE3

Electrocompetent BL21 DE3 were prepared according to the protocol. The following day, the LB Chlor plate was covered with colonies. Since BL21 are in contrast to BL21 pLys not ntaurally resistant to Chloramphenicol, they are obviously contaminated. In order to check whether this is also caused by an Indigoidine plasmid, extensive evaluation of stocks and transformed cells will be undertaken.

Test for Chloramphenicol Resistance

All stocks of competent cells were streaked on LB Chlor plates and incubated at 37°C ON.

Result

Cells Prepared Growth on Plate
E.coli DH10ß elec older no
E.coli DH10ß elec 21-09 no
E.coli BL21 DE3 chem original stock yes
E.coli BL21 DE3 elec older yes
E.coli BL21 DE3 elec 22-09 yes
E.coli BL21 pLys elec older yes

Thus, the BL21 DE3 we obtained most probably are BL21 DE3 pLys, too.

New BL21 DE3

We obtained new BL21 DE3 from the Russel Lab, which were obtained from NEB directly. Cells were inocculated in 10 ml LB and streaked on LB Chlor plates. The vial was frozen again at -80°C.

Test for Indigoidine Contamination A

All competent cells as well as important transformed cells and recent mini preps of possible DelH plasmids were tested for contamination with an Indigoidine plasmid (which carries chloramphenicol resistance).

Reagent Amount [µl]
Template 0.5 or colony
RB69 10 µM 1
VF2 10 µM 1
OneTaq Master Mix 5
ddH2O 2.5
Cycles Temperature [°C] Time [s]
1 95 300
35 95 30
53 30
72 90
1 72 600
1 12 inf

Result

Expected band: 540 bp

Fig.22.x Numerous samples were analyzed for contamination with Indigoidine.

The following samples show Indigoidine band:

Name Cells Plasmids Indigoidine band
1 E.coli BL21 DE3 original - no
2 E.coli BL21 DE3 older - no
3 E.coli BL21 DE3 22-09 - no
4 E.coli DH10ß 21-09 LB - no
5 miniprep DelH C5 yes
6 miniprep DelH C7 yes
7 miniprep DelH C12 yes
8 miniprep DelH G11 yes
9 miniprep DelH IIA6 yes
10 miniprep DelH IID2 yes
11 E.coli BL21 DE3 older DelCluster 25-1B + pIK8.6 no
12 E.coli BL21 DE3 older DelCluster 25-1C + pIK8.6 no
13 E.coli BL21 DE3 older DelCluster 25-2A + pIK8.6 no
14 E.coli BL21 DE3 older DelCluster 25-3A + pIK8.6 no
15 E.coli BL21 DE3 older DelCluster 25-3B + pIK8.6 no
16 E.coli BL21 DE3 older DelCluster 25-4 + pIK8.6 no
17 E.coli BL21 DE3 older DelCluster 25-5 + pIK8.6 no
18 E.coli BL21 pLys - no
19 E.coli BL21 pLys DelH C5 + pIK8.6 no
20 E.coli BL21 pLys DelH C7 + pIK8.6 no
21 E.coli BL21 pLys DelH C12 + pIK8.6 no
22 E.coli BL21 pLys DelH G11 + pIK8.6 no
23 E.coli BL21 pLys DelH IIA6 + pIK8.6 no
24 E.coli BL21 pLys DelH IID2 + pIK8.6 no
25 miniprep pIK2.6 yes
26 E.coli DH10ß white colony DelRest + DelH I6B + pIK2.6 no
27 E.coli DH10ß 21-09 ACM - no
+ miniprep pRB22.4 yes

All stocks are clean, only the DelH minipreps as well as the one from pIK2.6 are positive for Indigoidine. Therefore, we identified the source of contamination for the triple clone, which turned blue. We however cannot explain the positive result for the DelH minipreps, which obviously harbour a second plasmid.

Test for Indigoidine Contamination B

Again, all minipreps which were send for sequencing as well as a positive control a negative control and two recently prepared minipreps from other teams were tested for an Indigoidine contamination

Reagent Amount [µl]
Template 0.5 or colony
RB69 10 µM 1
VF2 10 µM 1
OneTaq Master Mix 5
ddH2O 2.5
Cycles Temperature [°C] Time [s]
1 95 300
35 95 30
53 30
72 90
1 72 600
1 12 inf

Result

Expected band: 540 bp

Fig.22.x Test for contamination with Indigoidine plasmid
Lane Cells originating from Miniprep Indigoidine band
upper row
2 E.coli BL21 DE3 22-09 DelH Plasmid I C5 23-09 yes
3 E.coli BL21 DE3 22-09 DelH Plasmid I C7 23-09 yes
4 E.coli BL21 DE3 22-09 DelH Plasmid I C12 23-09 yes
5 E.coli BL21 DE3 22-09 DelH Plasmid I G11 23-09 yes
6 E.coli BL21 DE3 22-09 DelH Plasmid II A6 23-09 yes
7 E.coli BL21 DE3 22-09 DelH Plasmid II D2 23-09 yes
8 E.coli BL21 DE3 22-09 DelH Plasmid III A9 23-09 yes
9 E.coli BL21 DE3 22-09 DelH Plasmid III D7 23-09 yes
10 E.coli BL21 DE3 22-09 DelH Plasmid III D9 23-09 yes
11 E.coli BL21 DE3 22-09 DelH Plasmid III D11 23-09 yes
12 E.coli BL21 DE3 22-09 DelH Plasmid III E6 23-09 yes
13 E.coli BL21 DE3 22-09 DelH Plasmid III E7 23-09 yes
14 E.coli DH10ß older DelH Plasmid III F6 23-09 yes
15 E.coli DH10ß older DelH Plasmid III F7 23-09 yes
16 E.coli DH10ß older DelH Plasmid III G3 23-09 yes
17 E.coli DH10ß older DelH Plasmid III G4 23-09 yes
18 E.coli DH10ß chem Indigoinide Plasmid pRB22 (positive) yes
lower row
2 E.coli DH10ß chem Indigoinide Plasmid pRB17 (negative) yes
3 E.coli DH10ß chem Indigoinide Plasmid recent miniprep K1 (negative) yes
4 E.coli DH10ß chem Indigoinide Plasmid recent miniprep K1 (negative) yes
5 E.coli DH10ß chem Indigoinide Plasmid pRB22 (positive) yes

Surprisingly, all samples show Indigoidine band at ~540 bp. There is a contamination and we have to identify the source!

Test for Indigoidine Contamination C

Again, all minipreps which were send for sequencing as well as positive and negative controls and recently prepared minipreps from other teams were tested for an Indigoidine contamination. using two different primer pairs. Additionally, fake minipreps were performed to identify DNA contamination of buffers.

Reagent Amount [µl]
Template 0.5 or colony
RB69 10 µM 1
VF2 10 µM 1
OneTaq Master Mix 5
ddH2O 2.5
Cycles Temperature [°C] Time [s]
1 95 300
35 95 30
53 30
72 90
1 72 600
1 12 inf
Reagent Amount [µl]
Template 0.5 or colony
KH05 10 µM 1
VR 10 µM 1
OneTaq Master Mix 5
ddH2O 2.5
Cycles Temperature [°C] Time [s]
1 95 300
35 95 30
53 30
72 90
1 72 600
1 12 inf

Result

Expected band: ~1 Kb for KH05 and 540 bp for RB69

Fig.22.x Testing for indigoidin Contamination uding KH05 and VR
Fig.22.x Testing for indigoidin Contamination uding RB69 and VF2
Lane Date Template Indigoidine band
KH05 and VR
2 24-09 (negative) Fake miniprep: all old no
3 24-09 (negative) Fake miniprep: P1 aliquot 20-09 no
4 24-09 (negative) Fake miniprep: fresh P1 no
5 24-09 (negative) Fake miniprep: fresh P2 no
6 24-09 (negative) Fake miniprep: P2 aliquot no
7 24-09 (negative) Fake miniprep: fresh S3 no
8 24-09 (negative) Fake miniprep: fresh isoprop no
9 21-07 (negative) pKH1 no
10 21-07 (negative) pKH1 + new polymerase yes
11 21-07 (negative) pSB3C5 + new polymerase yes
12 - pRB22 (positive) yes
13 23-09 (negative) DelH Plasmid I C7 23-09 yes
14 23-09 (negative) DelH Plasmid I C12 23-09 yes
15 23-09 (negative) DelH Plasmid I G11 23-09 yes
16 23-09 (negative) DelH Plasmid II A6 23-09 yes
17 23-09 (negative) DelH Plasmid II D2 23-09 yes
18 23-09 (negative) DelH Plasmid III F7 23-09 yes
19 23-09 (negative) DelH Plasmid III F9 23-09 yes
20 23-09 (negative) DelH Plasmid III G3 23-09 yes
21 23-09 (negative) DelH Plasmid III G4 23-09 yes
22 23-09 (negative) DelH Plasmid I C5 23-09 yes
23 E.coli DH10ß culture (negative) DelH Plasmid II A6 no
24 - - no
Lane Date Template Indigoidine band
RB69 and VF2
2 24-09 (negative) Fake miniprep: all old yes
3 24-09 (negative) Fake miniprep: P1 aliquot 20-09 yes
4 24-09 (negative) Fake miniprep: fresh P1 yes
5 24-09 (negative) Fake miniprep: fresh P2 yes
6 24-09 (negative) Fake miniprep: P2 aliquot yes
7 24-09 (negative) Fake miniprep: fresh S3 yes
8 24-09 (negative) Fake miniprep: fresh isoprop yes
9 04-08 (negative) 6 maxi HM no
10 15-09 (negative) pPW06 BCI no
11 19-09 (negative) ccdB 7 no
12 19-09 (negative) c.3 7 no
13 19-09 (negative) c.3 14.B no
14 21-07 (negative) pKH1 no
15 - (positive) pRB22 yes
16 - - no

Amplification with KH05 shows contamination of new polymerase vial (shorter band) as well of DelH minipreps. Interestingly, the tested culture is NOT contaminated.

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