Team:Heidelberg/Tyrocidine week20 interms

From 2013.igem.org

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

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

Restriction Digest

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

  • pPW06:
  • pPW09:
  • pPW10:
  • pPW11:

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