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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

300px 420px The PCR for fragment C was repeated after it was completely used in Gibson assemblies

Quantification-Gel

300px

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

COLPCR 09 11 12 Tyc-part.png Screening for the Tyrocidine-part with VF2 and PW14

COLPCR 09 11 12 Ind-part.png 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.

COLPCR 06 09 10 11 12 Tyc-part.png Screening for the Tyrocidine-part with VF2 and PW14]]

COLPCR 06 09 10 11 12 Ind-part.png 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:

EcoRI 06 10 11.png digest with EcoRI after 20 min on gel]]

EcoRI 06 10 11 LONG.png 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.