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Exeter/23 July 2013
From 2013.igem.org
Unfortunately, some of our liquid cultures from yesterday appear to have not worked.
Also, the liquid cultures for OmpR and the OmpR promoter were mislabelled, so we're uncertain which is which. However, when we run "OmpR A" on a gel, it should be very obvious which is which, as OmpR is 917bp and the OmpR promoter is 108bp.
| Part | Did it work? | Source |
|---|---|---|
| NP1 (CcaS + Terminator) | ✗ | Transformation plate |
| NP2 (promoter, RBS, FixJ + Terminator | ✗ | Transformation plate |
| NP3 (yellow pigment + Terminator) | ✗ | Transformation plate |
| NP4 (magenta pigment + Terminator) | ✗ | Transformation plate |
| NP7 (YF1 + Terminator) | ✓ | Transformation plate |
| NP8 (FixJ + Terminator) | ✓ | Transformation plate |
| Ligation control | ✗ | Transformation plate |
| Lambda inverter system | ✗ | Streak plate in fridge |
| T7 promoter | ✗ | Streak plate in fridge |
| Promoter + RBS (BBa_K608002) | ✓ | Streak plate in fridge |
| RBS (BBa_B0034) | ✓ | Streak plate in fridge |
| OmpR "A" | ✓ | Streak plate in fridge |
| OmpR "B" | ✗ | Streak plate in fridge |
| RFP control | ✓ | Transformation plate |
| RBS + cph8 (BBa_K592018) | ✓ | Transformation plate |
The working liquid cultures (NP7, NP8, Promoter + RBS, RBS, OmpR "A" and RBS + cph8) were MiniPrepped, then NanoDropped, then had their concentrations increased using the SureClean protocol from 17/7/13. Each culture was replicated twice.
| Part | NanoDrop data (ng/µl) | NanoDrop data after SureClean protocol (ng/µl) |
|---|---|---|
| Promoter and RBS #1 | 3.5 | 107.3 |
| Promoter and RBS #2 | 2.1 | 107.3 |
| NP8 #1 | 4.5 | 113.3 |
| NP8 #2 | 5.9 | 40.1 |
| RBS (BBa_B0034) #1 | 14.0 | 64.8 |
| RBS (BBa_B0034) #2 | 2.2 | Would not pellet, SureClean failed |
| NP7 #1 | 35.5 | SureClean not required |
| NP7 #2 | 29.3 | SureClean not required |
| OmpR "A" #1 | 2.1 | 110.5 |
| OmpR "A" #2 | 2.0 | 19.5 |
| RBS and cph8 #1 | 3.5 | 197.1 |
| RBS and cph8 #2 | 12.5 | Would not pellet, SureClean failed |
Digest
To ensure that our genes of interest are present, we took a small amount of each DNA solution, digested them with EcoRI and PstI to remove them from the plasmids, then ran them on a gel.
Each eppendorf contains:
- 12 µl distilled water
- 2 µl 10X FastDigest Buffer w/ Green Dye
- 0.5 µl EcoRI
- 0.5µl PstI
- 5 µl DNA
The samples digested were:
- RBS and cph8 #1
- RBS (BBa_B0034) #1
- OmpR "A" #1
- NP8 #1
- NP7 #1
- Promoter and RBS (BBa_K608002) #1
We also ran a digest of our original RBS + cph8 BioBrick (BBa_K322124), cutting one sample with EcoRI and PstI and another with EcoRI and SpeI. This is because we think there may be a PstI cut-site within the BioBrick, as when we sent it of for sequencing, the data that came back seemed much, much shorter than we were expecting.
All of our digests were run against a 1kb GeneRuler DNA ladder.
| Lane | Contents |
|---|---|
| 1 | Ladder |
| 2 | RBS and cph8 (BBa_K322124), cut with EcoRI and SpeI |
| 3 | RBS and cph8 (BBa_K322124), cut with EcoRI and PstI |
| 4 | RBS and cph8 (BBa_K592018) #1 |
| 5 | Promoter and RBS (BBa_K608002) #1 |
| 6 | NP7 #1 (YF1 + Terminator) |
| 7 | OmpR "A" #1 |
| 8 | RBS (BBa_B0034) #1 |
| 9 | NP8 #1 (FixJ + Terminator) |
| 10 | Ladder |
Unfortunately, the gel would not run properly. The ladders ran perfectly, but there were no bands present for the other DNA samples whatsoever. There are a variety of reasons why this may have happened, but in the end, it basically means we need to start afresh! Not at all frustrating...
Liquid cultures
We made liquid cultures of...
- CcaR (BBa_K592002)
- CcaS (BBa_K592001)
- RBS (BBa_B0034)
- Magenta pigment (BBa_K592012)
- Yellow pigment (BBa_K592010)
- OmpR promoter (BBa_R0082)
- OmpR (BBa_K098011)
- YF1 Blue Light Sensor (BBa_K592004)
- Lambda inverter system (BBa_Q04510)
- FixJ (BBa_K592005)
- FixJ promoter (a.k.a FixL) (BBa_K592006)
- Three replicates of promoter and RBS (BBa_K608002)
New Digest/Ligation options
After speaking with several academics and PhD students, we have been recommended various new "recipes" for our digestions/ligations. They mainly involve using amounts/concentrations of DNA, varying times in the thermocycler and cutting our BioBricks in different orders. Any new "recipes" will be clearly marked, but anyone used to the usual iGEM protocols should be able to see the differences!
Take me back to the notebook.
With many thanks to our sponsors:
