Team:Heidelberg/Templates/DelH week20

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09-09 - 15-09-13

Characterization of DelH Plasmid pHM04 30-08 Clones 4, 7, 15

Colony-PCR Conditions CP.W20.A

Because the sequenced DelH-colonies 4, 7, 15 had different kinds of mutations (Deletion of one basepair but also an insertion of a whole sequence part => results are shwon in week 19) we made a new screening PCR of 30 new picked colonies named 31-60 of the plate 2 of the electroporated cells realized in week 19.
There were 30 colonies screened.

Template	30x 1 PC of S-A
Expected length [bp]	663
Named	31-60
Primer fw 10 µM	30x 2 µl VF2
Primer rev 10 µM	30x 2 µl DN07
iTaq Polymeras (2x)	30x 10 µl
ddH₂O	30x 6 µl

Cycles	Temperature [°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.20.1 Colony PCR of Gibson assembled DelH-BB without mRFP (loaded 10 µL of PCR)
l1:50 bp ladder, l2-17:colonies 31-46
l4, l10,l11,l17:show the specific band at 663 bp

Fig.20.2 Colony PCR of Gibson assembled DelH-BB without mRFP (loaded 10 µL of PCR)
l1:50 bp ladder, l2-15:colonies 47-60, l16: ddH₂O
l6, l13:show the specific band at 663 bp

Colonies 33, 39, 40, 46, 51, 58 showed band.

=> Send in for sequencing after isopropanol ethanol purification.

Sequencing

The colonies 33, 39, 40, 46, 51, 58 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 add 2 µl DN07 primer (10µM).

Result

Colony	Alignment File	Conclusion
H33	sequencing insufficient
H39	sequencing insufficient
H40	sequencing insufficient
H46	sequencing insufficient
H51v	sequencing insufficient
H58	Sequencing of clone H58 with DN_07	insertion in the primer region of DelH-backbone (pHM04)

Colony-PCR Conditions CP.W20.B

New picked olonies 2x 95 well plate

Template	95x 1 µl of colony
Expected length [bp]	663
Named	I 1A - I 12H , II 1A - II 12H
Primer fw 10 µM	105x 2 µl VF2
Primer rev 10 µM	105x 2 µl DN07
iTaq Polymerase (2x)	105x 10 µl
ddH₂O	105x 5 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.20.3 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate I(loaded 10 µL of PCR)
l1:50 bp ladder, l2-l26:colonies 1A-4A

Fig.20.4 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate I (loaded 10 µL of PCR)
l1:50 bp ladder, l2-l26:colonies 4B-7B

Fig.20.5 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate I(loaded 10 µL of PCR)
l1:50 bp ladder, l2-l26:colonies 7C-9H

Fig.20.6 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate I (loaded 10 µL of PCR)
l1:50 bp ladder, l2-l26:colonies 10A-12H

Fig.20.7 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II (loaded 10 µL of PCR)
l1:50 bp ladder, l2-l26:colonies 1A-3G

Fig.20.8 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II (loaded 10 µL of PCR)
l1:50 bp ladder, l2-l24:colonies 3H-6F

Fig.20.9 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II (loaded 10 µL of PCR)
l2-l26:colonies 6G-9G

Fig.20.10 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II (loaded 10 µL of PCR)
l1:50 bp ladder, l2-l26:colonies 9H-12H

Colonies collected in table below show a definit result.

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

Colony of plate I	Colony of plate II
1F	2D
1G	2C
2A	3G
2H	4A
3B	4E
4C	7C
4E	7G
4F	8B
4H	10E
5E	10H
5F	11A
5G
(5H)
6B
6C
6D
7H
8B
8D
8F
8G
9B
9C
9F
10B
10C
10G
11F
12G

=> The next step is a test restriction digest with PvuI

Test Restriction Digest

We performed a test digest with PvuI-HF of the screened samples which showed a postive band at 663 bp in the screening PCR. Therefore we prepared a Master Mix with:

Enzyme	CutSmartBuffer [µl]	ddH₂O [µl]	Total amount [µl]
45x 0.5 µl = 22.5 µl	45x 2 µl = 90 µl	45x 16.5 µl = 742.5 µl	45x 19 µl = 855 µl

Incubation time: 1:20 h at 37 °C

Result

Expected bands: 11.5 Kb, 8.5 Kb and 2.6 Kb.

Fig.20.11. Test digest of pHM04 from different colonies with PvuI (loaded 20 µL of PCR)
l1:2log,l2:1F,l3: 1G,l4: 2A,l5: 3B,l6: 4C,l7: 4E,l8: 4F,l9: 4H,l10: 5E,l11: 5F,l12: 5G,l13: 5H

Fig.20.12. Test digest of pHM04 from different colonies with PvuI (loaded 20 µL of PCR)
l1:2log,l2:6B,l3: 6C,l4: 6D,l5: 7H,l6: 8B,l7:8D,l8: 8F,l9:8G,l10: 9B,l11: 9C,l12:9F,l13: 10C

Fig.20.13. Test digest of pHM04 from different colonies with PvuI (loaded 20 µL of PCR)
l1:2log,l2:plate I 10A,l3: 10B,l4: 10C,l5: 10D,l6: 10G,l7: 11F,l8: 12G,l9: plateII 2B,l10: 2D,l11: 2C,l12: 3G,l13: 4A

Fig.20.14. Test digest of pHM04 from different colonies with PvuI (loaded 20 µL of PCR)
l1:2log,l2:4E,l3: 7C,l4: 7G,l5: 8A,l6: 8B,l7:10E,l8: 10H,l9:11A

Some colonies showed the expected bands (see table below).

=> These are sent in for sequencing after a miniprep.

The table below shows the result of the restriction digest of different colonies containing the pHM04 plasmid. The restriction digest was positive if the expected bands were pesent.

Colony of plate I	Show expected bands	Colony of plate II	Show expected bands
1F	-	2D	-
1G	-	2C	-
2A	-	3G	+
2H	-	4A	-
3B	-	4E	+
4C	-	7C	-
4E	+	7G	+
4F	-	8B	+
4H	+	10E	-
5E	-	10H	-
5F	-	11A	-
5G	-	-	-
(5H)	-	-	-
6B	+	-	-
6C	+	-	-
6D	+	-	-
7H	+	-	-
8B	-	-	-
8D	-	-	-
8F	-	-	-
8G	+	-	-
9B	-	-	-
9C	-	-	-
9F	-	-	-
10B	+	-	-
10C	-	-	-
10G	+	-	-
11F	+	-	-
12G	-	-	-

Colony	Concentration ng/µl
I 4E	1597
I 4H	495
I 6B	255
I 6C	2445
I 6D	2101
I 7H	1539
I 8G	1589
I 10B	1359
I 10G	1863
I 11F	1886
II 3G	564
II 4E	2107
II 7G	1913
II 8B	955

Sequencing

Colony of plate I	Concentration ng/µl	Amount for sequencing [µl]	ddH₂O [µl]	Primer (DN07 [10 µM]) added [µl]
I 4E	1597	1	14	2
I 4H	495	1.5	13.5	2
I 6B	255	5	10	2
I 6C	2445	0.5	14.5	2
I 6D	2101	0.5	14.5	2
I 7H	1539	1	14	2
I 8G	1589	1	14	2

Result

Colony	Sequencing	Notes	Alignment File
I 4E	-	(Deletion of G in coding sequence)	Sequencing of clone I 4E with DN_07
I 4H	-	(Deletion of G in coding sequence)	Sequencing of clone I 4H with DN_07
I 6B	+ ?	a deletion in the RBS	Sequencing of clone I 6B with DN_07 Sequencing of clone I 6B with VF2
I 6C	-	(9 deletion)	Sequencing of clone I 6C with DN_07
I 6D	-	Deletion	Sequencing of clone I 6D with DN_07
I 7H	-	Deletion	Sequencing of clone I 7H with DN_07
I 8G	-	Deletion	Sequencing of clone I 8G with DN_07
I 8B	-	Deletion of G in coding sequence	discarded
I 10B	-	Deletion of G in coding sequence	discarded
I 10G	-	Insertion of G in coding sequence	discarded
I 11F	-	Deletion of G in coding sequence	discarded
II 3G	-	G Deletion of the ATG (start-codon) and 3 bp later deletion of C	discarded
II 4E	-	Deletion of G in coding sequence	discarded
II 7G	-	G Deletion of the ATG (start-codon) and 3 bp later deletion of C	discarded

Because the colony 6B is possibly positive, the next steps are:

1. SDS-PAGE

2. Sequencing over gibson-assembled parts

3. Triple electroporation with the plasmig of Del-rest and MalonylCoA (see lab book Delftibactin)

Amplification of Backbone pSB6A1

PCR Conditions BB.W20.A

Reagent	BB pSB6A1	BB pSB6A1
Expected length [Kb]	4.4	4.4
Template	0.5 µl pSB6A1 30-8	0.5 µl pSB6A1 30-8
Primer 10 µM fw	2 µl HM_17	2 µl HM_17
Primer 10 µM rev	2 µl FS_77	2 µl FS_77
Phusion Flash Master Mix (2x)	10 µl	10 µl
DMSO	1 µl	1 µl
ddH₂O	4.5 µl	4.5 µl

Cycles	Temperature [°C]	Time [s]
1	98	10
12	98	1
	68↓	5
	72	3:00 min
18	98	1
	66	5
	72	3:00 min
1	72	5:00 min
1	4	inf

Cycles	Temperature [°C]	Time [s]
1	98	10
30	98	1
30	72	3:00 min
1	72	5:00 min
1	4	inf

Result

Expected band: 4.4 Kb

Fig.20.15. Amplification of BB pSB6A1 with new primer and primer HM17 (loaded 20 µL of PCR)
l1:2 log ladder,l2:BB amplified HM17 and FS77 (68td),l3:BB amplified HM17 and HM20 (68td),l4: BB amplified HM17 and FS77 (2step),l5: BB amplified HM17 and HM20 (2step) - no yield

Very weak amplification, Primer-Dimers/Oligomers, carry over of Backbone pSB6A1 (including mRFP and therefore creating a double band, since template is 700bp longer than expected PCR product).

=> Repeat PCR with less stringent conditions to increase yield until unexpected bands appear or yield is high enough and reduce amount of template DNA (use 0.5 µL of a 1:10 delution)

=>Furthermore, elongation time was reduced, as template is only about 4.4 Kb long instead of the putative 7 Kb.

PCR Conditions BB.W20.B

Reagent	BB pSB6A1	BB pSB6A1
Expected length [Kb]	4.4	4.4
Template	0.5 µl pSB6A1 (3 ng/µL)	0.5 µl pSB6A1 (3 ng/µL)
Primer 10 µM fw	2 µl HM_17	2 µl HM_17
Primer 10 µM rev	2 µl FS_77	2 µl FS_77
Phusion Flash Master Mix (2x)	10 µl	10 µl
DMSO	1 µl	1 µl
ddH₂O	4.5 µl	4.5 µl

Cycles	Temperature [°C]	Time [s]
1	98	10
12	98	1
	66↓	5
	72	80
18	98	1
	64	5
	72	80
1	72	5:00 min
1	4	inf

Cycles	Temperature [°C]	Time [s]
1	98	10
30	98	1
30	58	5
72	80
1	72	5:00 min
1	4	inf

Result

Expected band: 4.4 Kb

Fig.20.16. Amplification of BB pSB6A1 with new primer and primer HM17 with different PCR conditions(loaded 20 µL of PCR)
l1:2 log ladder,l2:BB amplified HM17 and HM20 (66°C),l3:BB amplified HM17 and FS77 (66°C),l4:BB amplified HM17 and HM20 (58°C), l5:BB amplified HM17 and FS77 (58°C) - l4-5 show the expected band at 4.4 KB

Gel shows best amplification of fragment using annealing temperature at 58°C.

=> Repetition of PCR at a constant annealing temperature of 58°C.

PCR Conditions BB.W20.C

Performed 6x PCR with FS77 & 6x PCR with HM20, so we have enough yield for the Gibson assembly.

Reagent	BB pSB6A1	BB pSB6A1
Expected length [Kb]	4.4	4.4
Template	0.5 µl pSB6A1 (3 ng/µL)	0.5 µl pSB6A1 (3 ng/µL)
Primer 10 µM fw	2 µl HM_17	2 µl HM_17
Primer 10 µM rev	2 µl FS_77	2 µl FS_77
Phusion Flash Master Mix (2x)	10 µl	10 µl
DMSO	1 µl	1 µl
ddH₂O	4.5 µl	4.5 µl

Cycles	Temperature [°C]	Time [s]
1	98	10
30	98	1
	58	5
	72	80
1	72	5:00 min
1	4	inf

Result

Expected band: 4.4 Kb

Fig.20.17. Amplification of BB pSB6A1 with new primer and primer HM17 with a constant annealing temperature at 58°C(loaded 20 µL of PCR)
l1:2 log ladder,l2-7:BB amplified HM17 and FS77,l8-13:BB amplified HM17 and HM20 - no bands visible

Gel does not show the expected bands.

=> What happened? Why is it not reproducible? Run a PCR with other conditions.

PCR Conditions BB.W20.D

Reagent	BB pSB6A1	BB pSB6A1
Expected length [Kb]	4.4	4.4
Template	1 µl pSB6A1 (3 ng/µL)	1 µl pSB6A1 (3 ng/µL)
Primer 10 µM fw	2 µl HM_17	2 µl HM_17
Primer 10 µM rev	2 µl FS_77	2 µl FS_77
Phusion Flash Master Mix (2x)	10 µl	10 µl
ddH₂O	5.0 µl	5.0 µl

Cycles	Temperature [°C]	Time [s]
1	98	10
12	98	1
	60↓	5
	72	80
18	98	1
	68	5
	72	80
1	72	5:00 min
1	4	inf

Result

Expected band: 4.4. Kb

Fig.20.18. Amplification of BB pSB6A1 with new primer and primer HM17 with a td PCR (60°C) and a constant annealing temperature of 68°C(loaded 20 µL of PCR)
l1:2 log ladder,l2:BB amplified HM17 and FS77,l3:BB amplified HM17 and HM20 - no yield

Gel shows again no amplification of backbone.

=> Further optimize PCR conditions.

PCR Conditions BB.W20.E

Reagent	BB pSB6A1	BB pSB6A1
Expected length [Kb]	4.4	4.4
Template	0.5 µl pSB6A1 (3 ng/µL)	0.5 µl pSB6A1 (3 ng/µL)
Primer 10 µM fw	1 µl HM_17	1 µl HM_17
Primer 10 µM rev	1 µl FS_77	1 µl FS_77
Phusion Flash Master Mix (2x)	10 µl	10 µl
DMSO	1	1
ddH₂O	6.5 µl	6.5 µl

Cycles	Temperature [°C]	Time [s]
1	98	10
30	98	1
	58	5
	72	80
1	72	5:00 min
1	4	inf

Cycles	Temperature [°C]	Time [s]
1	98	10
12	98	1
	66↓	5
	72	1:20 min
18	98	1
	64	5
	72	80
1	72	5:00 min
1	4	inf

Result

Expected band: 4.4 Kb

Fig.20.19. Amplification of BB pSB6A1 with new primer and primer HM17 (loaded 20 µL of PCR)
l1:2 log ladder,l2:BB amplified HM17 and HM20 (66td),l3:BB amplified HM17 and FS77 (66td),l4: BB amplified HM17 and HM20 (58°C),l5: BB amplified HM17 and FS77 (58°C) - l4-5 show specific band - was cut out

Gel shows amplification using new HM17.

=> Fragments were cut and gel extracted.

PCR Conditions BB.W20.F

Run 6x PCR with HM20 and 6x PCR with FS77 for higher yield

Reagent	BB pSB6A1	BB pSB6A1
Expected length [Kb]	4.4	4.4
Template	0.5 µl pSB6A1 (3 ng/µL)	0.5 µl pSB6A1 (3 ng/µL)
Primer 10 µM fw	1 µl HM_17	1 µl HM_17
Primer 10 µM rev	1 µl FS_77	1 µl FS_77
Phusion Master Mix (2x)	10 µl	10 µl
DMSO	1	1
ddH₂O	6.5 µl	6.5 µl

Cycles	Temperature [°C]	Time
1	98	10
30	98	1
	58	5
	72	80
1	72	5:00 min
1	4	inf

The PCR was direcly digested with DpnI afterwards.

Restriction Digest with DpnI

10 of the 12 PCRs (5x FS & 5x HM) were cut with DpnI. There we used the exact PCR product as generated earlier.
Incubated at 37°C ON
The following table presents the amount for all reactions

Sample	Buffer	Enzyme	ddH₂O
10x 20 µl PCR	10x 2.5 µl CutSmart Buffer	2.5 µl DpnI	23 µl

Afterwards, a 0.8% gel was run for 1 h at 100 V and a gel-purification was performed with the Qiagen Gel Extraction Kit

Result

Expected band: 4.4 Kb

Fig.20.20. Restriction digested BB pSB6A1 with new primer and primer HM17 at an annealing temperature of 58°C (loaded 20 µL of PCR)
l1:2 log ladder,l2-7:BB amplified HM17 and FS77,l8-13:BB amplified HM17 and HM20 - specific band at 4 Kb

Fig.20.21. Restriction digested BB pSB6A1 with new primer and primer HM17 at an annealing temperature of 58°C (loaded 20 µL of PCR)
l1:2 log ladder,l2-7:BB amplified HM17 and FS77,l8-13:BB amplified HM17 and HM20 - specific band at 4 Kb

Gel shows expected bands.

=> Fragments were gel extracted.

Sample	DpnI digested	Concentration [ng/µl]
BB 16 (amplified with FS77)	yes	6.2
BB 16 (amplified with HM20)	yes	6.6

Generation of DelH Plasmid pHM04 15-09

Gibson Assembly

Fragment	Concentration [ng/µl]	Amount with BB16 (FS77) [µl]	Amount with BB16 (HM20) [µl]
DN11-FS66	172.5	0.78	0.81
FS67-FS68	120.7	0.95	0.99
FS69-FS70	211.3	0.94	0.98
FS71-HM08	146	0.55	0.57
BB16 amplified with FS77	6.2	6.78	-
BB16 amplified with HM20	6.6	-	6.64

Incubate for 1h at 50°C

Electroporation

Afterwards, 5 µl of each Gibson assembly were taken out and 10 µl ddH₂O was added
With 10 µl of the Gibson assembly, isopropanol purification was performed
6 different electroporations were performed (see scheme below)

Electroporation name	Inserted amount of Gibson Assembly	Plated out on agar plates
HM20 1	1 µl (Gibson + ddH₂O)	* 10 µl * rest
HM20 14	14 µl (Gibson + ddH₂O)	* 10 µl * rest
HM20 20	20 µl (isopropanol purified)	* 10 µl * rest
FS77 1	1 µl (Gibson + ddH₂O)	* 10 µl * rest
FS77 14	14 µl (Gibson + ddH₂O)	* 10 µl * rest
FS77 20	20 µl (isopropanol purified)	* 10 µl * rest

Team:Heidelberg/Templates/DelH week20

From 2013.igem.org

Contents

09-09 - 15-09-13

Characterization of DelH Plasmid pHM04 30-08 Clones 4, 7, 15

Colony-PCR Conditions CP.W20.A

Result

Sequencing

Result

Colony-PCR Conditions CP.W20.B

Result

Test Restriction Digest

Result

Sequencing

Result

Amplification of Backbone pSB6A1

PCR Conditions BB.W20.A

Result

PCR Conditions BB.W20.B

Result

PCR Conditions BB.W20.C

Result

PCR Conditions BB.W20.D

Result

PCR Conditions BB.W20.E

Result

PCR Conditions BB.W20.F

Restriction Digest with DpnI

Result

Generation of DelH Plasmid pHM04 15-09

Gibson Assembly

Electroporation