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Team:Heidelberg/Tyrocidine week20 interms
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(Created page with " == 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 F...") |
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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: | + | [[File:Heidelberg_Tyc-Ind Fusion_extended.jpeg|300px|center|thumb]] |
=== Amplifications === | === Amplifications === | ||
| Line 484: | Line 484: | ||
==== Gel-Pictures ==== | ==== Gel-Pictures ==== | ||
| - | [[File: | + | [[File:Heidelberg_TIFEXT_ABDEFGHJKLMN_PCR_1st.png|300px]] |
| - | [[File: | + | [[File:Heidelberg_TIFEXT_CCIIMMNN_PCR_2nd.png|420px]] |
| - | [[File: | + | [[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 ==== | ==== Quantification-Gel ==== | ||
| - | [[File: | + | [[File:Heidelberg_TIFEXT_all_fragments_QUANT_1st.png|300px]] |
=== Gibson Assembly === | === Gibson Assembly === | ||
| Line 596: | Line 596: | ||
==== Colony-PCR ==== | ==== Colony-PCR ==== | ||
<gallery> | <gallery> | ||
| - | File: | + | File:Heidelberg_COLPCR_09_11_12_Tyc-part.png|Screening for the Tyrocidine-part with VF2 and PW14 |
| - | File: | + | File:Heidelberg_COLPCR_09_11_12_Ind-part.png|Screening for the Indigoidine-part with VR and KH05 |
</gallery> | </gallery> | ||
| Line 603: | Line 603: | ||
<gallery> | <gallery> | ||
| - | File: | + | File:Heidelberg_COLPCR_06_09_10_11_12_Tyc-part.png|Screening for the Tyrocidine-part with VF2 and PW14]] |
| - | File: | + | File:Heidelberg_COLPCR_06_09_10_11_12_Ind-part.png|Screening for the Indigoidine-part with VR and KH05]] |
</gallery> | </gallery> | ||
| Line 615: | Line 615: | ||
<gallery> | <gallery> | ||
| - | File: | + | File:Heidelberg_EcoRI_06_10_11.png|digest with EcoRI after 20 min on gel]] |
| - | File: | + | File:Heidelberg_EcoRI_06_10_11_LONG.png|digest with EcoRI after 40 min on gel]] |
</gallery> | </gallery> | ||
After only 20 minutes of running, the bands were not yet clearly separated. Waiting for another 20 minutes lead to a interpretable result. | After only 20 minutes of running, the bands were not yet clearly separated. Waiting for another 20 minutes lead to a interpretable result. | ||
Latest revision as of 19:11, 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:
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.
