Team:Heidelberg/Delftibactin/DelRest

From 2013.igem.org

(Difference between revisions)

Revision as of 02:58, 30 September 2013

Del Rest. Creating a 31 kbp plasmid.

Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.

Week 1

At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed. After the Transformation to DH10β cells and screening by restriction digest we could send samples for the Dipeptide and Tetrapeptide I to sequencing and obtained a positive alignment. Hence we transformed BAP I cells with the positive constructs. The same was done earlier that week with the Tripeptide I - NRPS. After a second round of picking and restriction digest, positive colonies were also found for the missing constructs and DNA was sent to sequencing. If the sequencing turns out to be correct, we can transform BAP I cells with the missing constructs, and can either way start with the preparation for mass-spec. Besides that, primers for the Tyrocidine-Indigoidine-Fusion-Project were designed and ordered.

Week 2

Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.

At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed. After the Transformation to DH10β cells and screening by restriction digest we could send samples for the Dipeptide and Tetrapeptide I to sequencing and obtained a positive alignment. Hence we transformed BAP I cells with the positive constructs. The same was...

Week 3

Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.

At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed. After the Transformation to DH10β cells and screening by restriction digest we could send samples for the Dipeptide and Tetrapeptide I to sequencing and obtained a positive alignment. Hence we transformed BAP I cells with the positive constructs. The same was...

Week 4

Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.

Week 5

At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed.

Week 6

Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.

Week 7

Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.

Week 8

Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.

Week 9

Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.

Methods:

Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. .

06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

PCR for amplification of del fragments E-G and A-E, AE+=PCR AE with DMSO, AE-=PCR without DMSO, M= Marker; wanted amplicon sizes are: AE=5.3kb, EG=11.0

Reaction

what	µL
D. acidovorans DSM-39	1
FS_02: (1/10)	1
FS_03: (1/10)	1
Phusion Master Mix	10
DMSO	1/-
dd H₂O	6/7

PCR for amplification of del fragments AE and EG, cut

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	5
12	98	1
	66 ↓ 0.5	5
	72	2:30 min
18	98	1
	63	5
	72	2:30 min
1	72	10 min
1	4	inf

Results:

Amplification worked with 5% DMSO
Repeat Amplification with the same protocol to increase concentration when DNA is extracted from gel slices

03-07-2013

Amplification from FS_02 to FS_05; 11.2 kb

Amplification of DelA-F and DelF-G, 03-07-13; run at 130 V, 0.8 % gel (TAE)

Gel after removing the DelA-F-fragment, 03-07-13.

Reaction

µL 1st PCR	what	µL 2nd PCR
1	D. acidovorans DSM-39	1
2.5	FS_02 (1/10)	2.5
2.5	FS_05 (1/10)	2.5
25	Phusion Master Mix	25
-	DMSO	2.5
19	dd H₂O	19

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	10
12	98	1
	68 ↓ 0.5	5
	72	3 min
18	98	1
	66	5
	72	3 min
1	72	10 min
1	4	inf

Results:

Amplification of DelAE worked

bands were cut out and DNA purified using QIAquick Gel Extraction Kit

sample has to be purified before further use since contamination with propanol was present

04-07-2013

Amplification from FS_02 to FS_05; 11.2 kb

Gel electrophoresis of the PCR products of DelA-F (first three lane (counted from left)) and DelF-G (4th till 6th lane), 04-07-2013

Gel after removing DelA-F-fragment, 04-07-2013

Reaction

what	µL 2nd PCR
D. acidovorans DSM-39	1
FS_02 (1/10)	2.5
FS_05 (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	10
12	98	1
	68 ↓ 0.5	5
	72	3 min
18	98	1
	66	5
	72	3 min
1	72	10 min
1	4	inf

Results:

Amplification of DelAE worked

bands were cut out and DNA purified using QIAquick Gel Extraction Kit resulting in a final concentratio of 10ng/µL

PCR will be repeated and gel slices purified with QIAX II Gel Extraction Kit, which is specifically designed to deliver higher yields when purifying fragments with sizes over 10 kbp

05-08-2013

Amplification from FS_02 to FS_07; 16.4 kb

Gel electrophoresis of DelA-G

Reaction

what	µl
D. acidovorans DSM-39	1
FS_02: (1/10)	1
FS_07: (1/10)	1
Phusion Master Mix	10
DMSO	1
dd H₂O	6

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	10
6	98	1
	66 ↓ 0.5	5
	72	5min 40s
24	98	1
	60	5
	72	5min 40s
1	72	17 min
1	4	inf

Results:

amplification of DelAG did not work
repeat PCR with higher annealing temperature to increase specifity

06-07-2013

Amplification from FS_04 to FS_07; 11.1 kb

PCR for amplification of del fragments E-G and A-E, AE+=PCR AE with DMSO, AE-=PCR without DMSO, M= Marker; wanted amplicon sizes are: AE=5.3kbp, EG=11.0

PCR for amplification of del fragments AE and EG, cut

Reaction

what	µl
D. acidovorans DSM-39	1
FS_04: (1/10)	1
FS_07: (1/10)	1
Phusion Master Mix	10
DMSO	1/-
dd H₂O	6/7

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	10
12	98	1
	68 ↓ 0.5	5
	72	3 min
18	98	1
	66	5
	72	3 min
1	72	10 min
1	4	inf

Results:

Amplification of DelEG worked but another unexpected product was amplified as well

band was cut out avoiding contamination with other product and DNA purified using QIAquick Gel Extraction Kit

05-07-2013

Amplification from FS_08 to FS_11; 6.5 kb

Gel electrophoresis of DelF-G, Backbone, DelF-G (5.1 kb), DelG (6.4 kb).

after Gel excision

Reaction

what	µl
D. acidovorans DSM-39	1
FS_08: (1/10)	1
FS_11: (1/10)	1
Phusion Master Mix	10
dd H₂O	6
DMSO	1

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	5
14	98	1
	62 ↓ 0.5	5
	72	3:20 min
16	98	1
	60	5
	72	3:20 min
1	72	12 min
1	4	inf

Results:

A weak band was visible, but it was on the wrong height.
Accidently the band was cut anyway.
Either the primers did not bind or the DNA still had to many secondary structures --> the consequence is to change the annealing temperature.

07-07-2013

Amplification from FS_08 to FS_11; 6.5 kb

PCR for amplification of del fragments DelAE (08.07), pSB4K5 (08.07) and DelG (07.07); lane1=Marker, lanes2-4=DelAE (5.3kbp), lanes5-10=pSB4K5 (4.2kbp), lanes11-13:DelG (6.4kbp); run at 100 V, 0.8 % gel (TAE)

PCR for amplification of del fragments DelAE (08.07), pSB4K5 (08.07) and DelG (07.07), cut

Reaction

what	µl
D. acidovorans DSM-39	1
FS_08: (1/10)	2.5
FS_11: (1/10)	2.5
Phusion Master Mix	25
dd H₂O	19
DMSO	2.5

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	5
12	98	1
	66 ↓ 0.5	5
	72	2:30 min
18	98	1
	63	5
	72	2:30 min
1	72	10 min
1	4	inf

Results:

There was no band visible on the gel.
Either the primers did not bind or the DNA still had to many secondary structures --> the consequence is to change the annealing temperature.

04-07-2013

Amplification from FS_12 to FS_13; 2.7 kb

Gel eletrophoresis of PCRs of DelF-G, DelO-P and DelL

Reaction

what	µl
D. acidovorans DSM-39	1
FS_12: (1/10)	1
FS_13: (1/10)	1
Phusion Master Mix	10
DMSO	1
dd H₂O	6

Conditions

Biorad MyCycler
Cycles	temperature [°C]	Time [s]
1	98	10
30	98	1
30	72	40
1	72	420
1	4	inf

Results:

Amplification of DelOP did not work
Amplification will be repeated to exclude that pipetting errors were the reason for the failure

04-07-2013

Amplification from FS_14 to FS_15; 1.4 kb

Gel eletrophoresis of PCRs of DelF-G, DelO-P and DelL

Reaction

what	µl
D. acidovorans DSM-39	1
FS_14: (1/10)	1
FS_15: (1/10)	1
Phusion Master Mix	10
DMSO	1
dd H₂O	6

Conditions

Biorad MyCycler
Cycles	temperature [°C]	Time [s]
1	98	10
30	98	1
30	72	40
1	72	7 min
1	4	inf

Results:

Amplification worked very well, we had a bright band on the right height.
Band was cut out and DNA purified using QIAquick Gel Extration Kit.

06-07-2013

Amplification from FS_14 to FS_15; 1.4 kb

PCR for amplification of del fragment L, L+=PCR delL with DMSO, L-=PCR without DMSO, M= Marker run at 135 V, 0.8 % gel (TAE); wanted amplicon size is: 1.4 kbp

Reaction

what	µl
D. acidovorans DSM-39	1
FS_14: (1/10)	1
FS_15: (1/10)	1
Phusion Master Mix	10
DMSO	1/-
dd H₂O	6/7

PCR for amplification of del fragment L, cut

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	10
12	98	1
	66 ↓ 0.5	5
	72	42
18	98	1
	63	5
	72	42
1	72	12 min
1	4	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.

05-07-2013

Amplification from FS_01 to FS_16; 4.2 kb

Gel electrophoresis of DelF-G, Backbone, DelF-G (5.1 kb), DelG (6.4 kb).

after Gel excision

Reaction

what	µl
Template pSB4K5	1
FS_16: (1/10)	1
FS_01: (1/10)	1
Phusion Master Mix	10
dd H₂O	6
DMSO	1

Conditions

Biorad MyCycler
Cycles-PCR	temperature [°C]	Time [s]
1	98	5
14	98	1
	62 ↓ 0.5	5
	72	3:20 min
16	98	1
	60	5
	72	3:20 min
1	72	12 min
1	4	inf

Results:

Amplification worked well and band was at expected height. Nevertheless the gel band did not run properly on the gel.

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