Team:Heidelberg/Tyrocidine week20 interms

From 2013.igem.org

(Difference between revisions)
Line 1: Line 1:
-
 
== Tyrocidine-Indigoidine fusion - extended ==
== 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:
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:Heidelberg_Tyc-Ind Fusion_extended.jpeg|1000px|center|thumb]]
+
[[File:Heidelberg_Tyc-Ind Fusion_extended.jpeg|300px|center|thumb]]
=== Amplifications ===
=== Amplifications ===

Revision as of 17:32, 4 October 2013

Contents

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:

Heidelberg Tyc-Ind Fusion extended.jpeg

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

Heidelberg TIFEXT ABDEFGHJKLMN PCR 1st.png Heidelberg TIFEXT CCIIMMNN PCR 2nd.png The PCR for fragment C was repeated after it was completely used in Gibson assemblies

Quantification-Gel

Heidelberg TIFEXT all fragments QUANT 1st.png

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.

Retrieved from "http://2013.igem.org/Team:Heidelberg/Tyrocidine_week20_interms"