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Team:Heidelberg/Tyrocidine week21 interms
From 2013.igem.org
ccdb Construct
Amplification
ccdb-C(tycC2)-indC plasmid (pJS01) Amplification of C(tycC2), ccdB and pSB1C3 DNA fragments (indC was stored in the fridge from former experiments).
- ccdB pcr contents
| What | µl |
|---|---|
| primer_fw (PW38) | 2 |
| primer_rv (PW41) | 2 |
| template (Gateway pDONR vector) | 2 |
| Phusion Flash 2x Master Mix | 10 |
| ddH20 | 4 |
- PCR-Program ccdB sequence
| Cycles | Temperature [°C] | Time [min:s] |
|---|---|---|
| 1 | 98 | 0:10 |
| 8 | 98 | 0:01 |
| 70 ↓0.5 | 0:05 | |
| 72 | 0:15 | |
| 28 | 98 | 0:01 |
| 65 | 0:05 | |
| 72 | 0:15 | |
| 1 | 72 | 1:30 |
| 1 | 10 | inf |
- pSB1C3 PCR contents
| wWhat | µl |
|---|---|
| primer_fw (PW23) | 2 |
| primer_rv (PW42) | 2 |
| template (pSB1C3) | 0.5 |
| Phusion Flash 2x Master Mix | 10 |
| ddH20 | 5.5 |
- PCR-Program pSB1C3 sequence
| Cycles | Temperature [°C] | Time [min:s] |
|---|---|---|
| 1 | 98 | 2:00 |
| 35 | 98 | 0:05 |
| 60 | 0:20 | |
| 72 | 1:00 | |
| 1 | 72 | 10:00 |
| 1 | 10 | inf |
- C(TycC2) PCR contents
| What | µl |
|---|---|
| primer_fw(PW21) | 2 |
| primer_rv(PW14) | 2 |
| template (B. Parabrevis) | 1 |
| Phusion Flash 2x Master Mix | 10 |
| ddH20 | 5 |
- PCR-Program C(TycC2)
| Cycles | Temperature [°C] | Time [min:s] |
|---|---|---|
| 1 | 98 | 2:00 |
| 35 | 98 | 0:01 |
| 59 | 0:05 | |
| 72 | 1:00 | |
| 1 | 72 | 5:00 |
| 1 | 10 | inf |
After amplification, an analytical gel electrophoresis was run to quantify DNA concentrations.
- C(TycC2) PCR contents
| What | Size [bp] | Estimated concentration |
|---|---|---|
| pSB1C3 | 2500 | 35 |
| ccdB | 600 | 30 |
| C(TycC2) | 1000 | 30 |
| indC | 4000 | 100 |
Assembly
Gibson assembly was followed by purification with isopropanol and transformation by heat shock into ccdB resistent and non-resistent (Top10) cells.
After 10 hours of culture growth, colony PCRs and test digests with Pst1 and EcoRI were conducted. Afterwards, minipreps of cultures containing the plasmids and concentration measurements were performed accompanied by test digests. Finally, positive constructs were transformed again and prepared for sequencing.
20130918 COLPCR ccdB-Ind labeled.png
Colony PCRs. On the left: ccdB (all positive except 8). On the right: Indigoidine (all positive). |
20130919 1kb ccdb-indc1-5 digest labeled.png
Digests of ccdB-indC constructs 1-5 with Pst1 and EcoRI. |
20130919 1kb ccdbIndc constructs5-6 digest labeled.png
Digests of ccdB-indC constructs 6-10 with Pst1 and EcoRI. |
20130919 1kb digest ccdbind labeled.png
Digests of ccdB-indC3 and 7 with Pst1 and EcoRI. Results were positive. |
- ccdB nanodrop measurements
| Colony | Concentration [ng/µl] |
|---|---|
| ccdB1 | 863.5 |
| ccdB2 | 436.9 |
| ccdB3 | 1258.5 |
| ccdB4 | 260.5 |
| ccdB5 | 106.6 |
| ccdB6 | 1064.4 |
| ccdB7 | 1213.0 |
| ccdB8 | 1301.4 |
| 3B | 982.3 |
| 3*B | 982.4 |
Sequencing results of ccdB3 and ccdB7 (just partwise sequencing) were positive. Biobrick can be submitted.
_C(TycC2)Q5_6B_15_QUANT_caption.png){kind=link}