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Contents 1 23-09 - 29-09-13 1.1 Mutagenesis of DelH Clone I 6B 1.1.1 Screening-PCR Conditions CP.W22.A 1.1.2 Result 1.1.3 Midiprep of Clones 2,3,4,7,8 and 10 1.1.4 Test restriction digest of Midipreps with MfeI and XbaI 1.1.5 Sequencing 1.1.6 Result 1.2 SDS-PAGE of Clone C5 1.2.1 Lysis of Cells 1.2.2 Electrophoresis 1.2.3 Staining and Destaining Procedure 1.2.4 Result 1.3 Generation of DelH Plasmid pHM04 18-09 1.3.1 Colony-PCR CP.W22.B 1.3.2 Result 1.3.3 Test Restriction Digest 1.3.4 Result 1.4 Generation of DelH Plasmid pHM04 20-09 1.4.1 Colony-PCR CP.W22.C 1.4.2 Result 1.4.3 Test Restriction Digest 1.4.3.1 Result 1.4.4 Sequencing 1.4.5 Result 1.5 Contamination of Cells with Chloramphenicol Resistance 1.5.1 Preparation of Electrocompetent BL21 DE3 1.5.2 Test for Chloramphenicol Resistance 1.5.3 Result 1.5.4 New BL21 DE3 1.5.5 Test for Indigoidine Contamination A 1.5.6 Result 1.5.7 Test for Indigoidine Contamination B 1.5.8 Result 1.5.9 Test for Indigoidine Contamination C 1.5.10 Result

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)

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

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

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

• 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

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

• 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

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	File:Heidelberg Sequencing Result pHM04 VF2 colony DH10b F7.clustal.txt
III F9	File:Heidelberg Sequencing Result pHM04 VF2 colony DH10b F9.clustal.txt
III G3	File:Heidelberg Sequencing Result pHM04 VF2 colony DH10b G3.clustal.txt
III G4	File:Heidelberg Sequencing Result pHM04 VF2 colony DH10b G4.clustal.txt

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

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

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

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.