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From 2013.igem.org

Contents 1 Tyrocidine-Indigoidine fusion - extended 1.1 Amplifications 1.1.1 Protocols 1.1.1.1 A 1.1.1.2 B 1.1.1.3 C 1.1.1.4 D 1.1.1.5 E 1.1.1.6 F 1.1.1.7 G 1.1.1.8 H 1.1.1.9 I 1.1.1.10 J 1.1.1.11 K 1.1.1.12 L 1.1.1.13 M 1.1.1.14 N 1.1.2 Gel-Pictures 1.1.3 Quantification-Gel 1.2 Gibson Assembly 1.2.1 pPW06 (Orn-Val-Ind) 1.2.2 pPW07 (Orn-Val-Val-Ind) 1.2.3 pPW08 (Val-Val-Ind) 1.2.4 pPW09 (Pro-Leu-Ind) 1.2.5 pPW10 (Pro-Leu-Val-Ind) 1.2.6 pPW11 (Phe-Orn-Leu-Ind) 1.2.7 pPW12 (Phe-Orn-Leu-Val-Ind) 1.3 Screening for positive transformants 1.3.1 Colony-PCR 1.3.2 Restriction Digest

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.