Team:Heidelberg/Tyrocidine week20 interms

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Revision as of 17:08, 4 October 2013

Tyrocidine-Indigoidine fusion - extended

As Valin-Indigoidine and its synthetase could be detected by TLC and SDS-PAGE respectively, we decided to increase the number of Fusion-constructs in order to test Indigoidine as possible tag for NRPs and their synthesis. Herefore, we aim to build the following 7 NRPs:

Amplifications

Protocols

A

what	µl
pIK04	1
PW26	2
PW27	2
Phusion Flash 2x Master Mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	0:05
35	98	0:05
	66	0:10
	72	0:50
1	72	10:00
1	10	inf

B

what	µl
B. parabrevis	1
PW28	2
PW29	2
Phusion Flash 2x Master Mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	0:05
35	98	0:05
	58	0:10
	72	0:30
1	72	10:00
1	10	inf

C

what	µl
pPW05	1
PW30	2
PW16	2
Phusion Flash 2x Master Mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	0:05
35	98	0:05
	64	0:10
	72	2:00
1	72	10:00
1	10	inf

D

what	µl
pIK04	1
PW37	2
PW23	2
Phusion Flash 2x Master Mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	0:05
35	98	0:05
	66	0:10
	72	0:50
1	72	10:00
1	10	inf

E

what	µl
B. parabrevis	1
PW28	2
PW31	2
Phusion Flash 2x Master Mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	0:05
35	98	0:05
	63	0:10
	72	1:00
1	72	10:00
1	10	inf

F

what	µl
B. parabrevis	1
PW32	2
PW29	2
Phusion Flash 2x Master Mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	0:05
35	98	0:05
	58	0:10
	72	0:30
1	72	10:00
1	10	inf

G

what	µl
pPW05	1
PW19	2
PW31	2
Phusion Flash 2x Master Mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	0:05
35	98	0:05
	66	0:10
	72	0:50
1	72	10:00
1	10	inf

H

what	µl
pIK03	1
IK13	2
PW33	2
Phusion Flash 2x Master Mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	0:05
35	98	0:05
	65	0:10
	72	1:40
1	72	10:00
1	10	inf

I

what	µl
pPW05	1
PW34	2
PW16	2
Phusion Flash 2x Master Mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	0:05
35	98	0:05
	60	0:10
	72	1:40
1	72	10:00
1	10	inf

J

what	µl
pIK04	1
IK23	2
PW23	2
Phusion Flash 2x Master Mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	0:05
35	98	0:05
	66	0:10
	72	0:50
1	72	10:00
1	10	inf

K

what	µl
pIK03	1
IK13	2
PW35	2
Phusion Flash 2x Master Mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	0:05
35	98	0:05
	65	0:10
	72	1:40
1	72	10:00
1	10	inf

L

what	µl
B. parabrevis	1
PW36	2
PW29	2
Phusion Flash 2x Master Mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	0:05
35	98	0:05
	58	0:10
	72	0:30
1	72	10:00
1	10	inf

M

what	µl
pIK04	1
IK16	2
PW33	2
Phusion Flash 2x Master Mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	0:05
35	98	0:05
	65	0:10
	72	3:10
1	72	10:00
1	10	inf

N

what	µl
pIK04	1
IK16	2
PW35	2
Phusion Flash 2x Master Mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	0:05
35	98	0:05
	65	0:10
	72	3:10
1	72	10:00
1	10	inf

Gel-Pictures

Quantification-Gel

Gibson Assembly

pPW06 (Orn-Val-Ind)

fragment	concentration [ng/µl]	volume for gibson assembly [µL]
A	40	1.61
B	35	0.92
C	30	6.97
D	40	0.50

pPW07 (Orn-Val-Val-Ind)

fragment	concentration [ng/µl]	volume for gibson assembly [µL]
A	40	1.16
C	30	4.32
D	40	0.36
E	20	3.49
F	35	0.66

pPW08 (Val-Val-Ind)

fragment	concentration [ng/µl]	volume for gibson assembly [µL]
C	30	6.07
F	35	0.93
G	40	1.63
OldG	15	1.36

pPW09 (Pro-Leu-Ind)

fragment	concentration [ng/µl]	volume for gibson assembly [µL]
H	20	6.35
I	40	3.02
J	25	0.63

pPW10 (Pro-Leu-Val-Ind)

fragment	concentration [ng/µl]	volume for gibson assembly [µL]
C	30	4.58
J	20	0.66
K	25	4.23
L	40	0.53

pPW11 (Phe-Orn-Leu-Ind)

fragment	concentration [ng/µl]	volume for gibson assembly [µL]
I	40	0.58
J	20	0.15
M	5	9.27

pPW12 (Phe-Orn-Leu-Val-Ind)

fragment	concentration [ng/µl]	volume for gibson assembly [µL]
C	30	1.77
J	20	0.26
L	40	0.20
N	10	7.77

Cells were transformed with 1 µl and 14 µl 1:2 diluted Gibson-Mix.

Screening for positive transformants

Colony-PCR

Screening for the Tyrocidine-part with VF2 and PW14

Screening for the Indigoidine-part with VR and KH05

First, there were colonies visible for the transformants of pPW09, pPW11 and pPW12. Later colonies for pPW06 and pPW10 followed.

Screening for the Tyrocidine-part with VF2 and PW14]]

Screening for the Indigoidine-part with VR and KH05]]

Restriction Digest

Positive samples were digested with restriction digest with EcoRI. The expected sizes of bands were:

pPW06:
pPW09:
pPW10:
pPW11:

digest with EcoRI after 20 min on gel]]

digest with EcoRI after 40 min on gel]]

After only 20 minutes of running, the bands were not yet clearly separated. Waiting for another 20 minutes lead to a interpretable result.

@@ Line 3: / Line 3: @@
 As Valin-Indigoidine and its synthetase could be detected by TLC and SDS-PAGE respectively, we decided to increase the number of Fusion-constructs in order to test Indigoidine as possible tag for NRPs and their synthesis. Herefore, we aim to build the following 7 NRPs:
-[[File:Tyc-Ind Fusion_extended.jpeg|1000px|center|thumb]]
+[[File:Heidelberg_Tyc-Ind Fusion_extended.jpeg|1000px|center|thumb]]
 === Amplifications ===
@@ Line 484: / Line 484: @@
 ==== Gel-Pictures ====
-[[File:TIFEXT_ABDEFGHJKLMN_PCR_1st.png|300px]]
+[[File:Heidelberg_TIFEXT_ABDEFGHJKLMN_PCR_1st.png|300px]]
-[[File:TIFEXT_CCIIMMNN_PCR_2nd.png|420px]]
+[[File:Heidelberg_TIFEXT_CCIIMMNN_PCR_2nd.png|420px]]
-[[File:TIFEXT_CC_PCR_3rd.png|190px|The PCR for fragment C was repeated after it was completely used in Gibson assemblies]]
+[[File:Heidelberg_TIFEXT_CC_PCR_3rd.png|190px|The PCR for fragment C was repeated after it was completely used in Gibson assemblies]]
 ==== Quantification-Gel ====
-[[File:TIFEXT_all_fragments_QUANT_1st.png|300px]]
+[[File:Heidelberg_TIFEXT_all_fragments_QUANT_1st.png|300px]]
 === Gibson Assembly ===
@@ Line 596: / Line 596: @@
 ==== Colony-PCR ====
 <gallery>
-File:COLPCR_09_11_12_Tyc-part.png|Screening for the Tyrocidine-part with VF2 and PW14
+File:Heidelberg_COLPCR_09_11_12_Tyc-part.png|Screening for the Tyrocidine-part with VF2 and PW14
-File:COLPCR_09_11_12_Ind-part.png|Screening for the Indigoidine-part with VR and KH05
+File:Heidelberg_COLPCR_09_11_12_Ind-part.png|Screening for the Indigoidine-part with VR and KH05
 </gallery>
@@ Line 603: / Line 603: @@
 <gallery>
-File:COLPCR_06_09_10_11_12_Tyc-part.png|Screening for the Tyrocidine-part with VF2 and PW14]]
+File:Heidelberg_COLPCR_06_09_10_11_12_Tyc-part.png|Screening for the Tyrocidine-part with VF2 and PW14]]
-File:COLPCR_06_09_10_11_12_Ind-part.png|Screening for the Indigoidine-part with VR and KH05]]
+File:Heidelberg_COLPCR_06_09_10_11_12_Ind-part.png|Screening for the Indigoidine-part with VR and KH05]]
 </gallery>
@@ Line 615: / Line 615: @@
 <gallery>
-File:EcoRI_06_10_11.png|digest with EcoRI after 20 min on gel]]
+File:Heidelberg_EcoRI_06_10_11.png|digest with EcoRI after 20 min on gel]]
-File:EcoRI_06_10_11_LONG.png|digest with EcoRI after 40 min on gel]]
+File:Heidelberg_EcoRI_06_10_11_LONG.png|digest with EcoRI after 40 min on gel]]
 </gallery>
 After only 20 minutes of running, the bands were not yet clearly separated. Waiting for another 20 minutes lead to a interpretable result.