Exeter/23 July 2013

From 2013.igem.org

Exeter iGEM 2013 · Paint by Coli

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


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.

Exeter iGEM 2013 · Paint by Coli