Team:UCC Ireland/Project/Notebook/24/07

Monday-24/07/17

Ligations from last week worked!

  • AmilCP blue colonies present
  • mCherry red colonies present
  • No ligase control of AmilCP

Ran repeat of K145279 experiment:

  • GFP under control of pTet and TetR.
  • OD600 - 0.799 @ T=0 of Cells.
  • Ran same six concentrations 2.5µg/L → 0.005µg/L verses blank LB versus a constitutively expressed GFP (BBa_K84001). Also included was a negative control of cells that did not express GFP so we used an overnight of cells for (BBa_K145201).

Experiment:

Ligation of TetR with Promoter-RFP using the Standard Assembly method. This vector will be tested with various concentrations of tetracycline and its fluorescence measured.

  • Plasmid prep of the samples:
    • TetR (BBa_K145201) : 113ng/microL
    • Promoter.RFP (BBa_I13521) : 50ng/microL
  • Restriction digest:
    • TetR with EcoR1, Spe1
    • Pro.RFP with EcoR1, Xba1
  • Gel extraction of TetR gene and gel digestion.
  • Measured the TetR gene concentration. Low yield of 4.3ng/microL obtained. Sample not workable and quite contaminated. Gel extraction will be repeated tomorrow.

Tuesday-25/07/17

Experiment from Monday repeated - Restriction digest of TetR and ran on a gel and excised. Yield of DNA and purity measured using the nanodrop. - Low yield of 6.6ng/microL.

Ligation of TetR + Pro-RFP. Transformation of the ligated plasmid.

Plasmid preps of the overnights : AmilCP and mCherry.


Wednesday-26/07/17

-Gel extraction of TetR gene - try to increase purity and yield. Methods to increase yield of DNA from gel extraction:

  • Wash DNA with ethanol multiple times - to remove salt residues
  • Make sure all ethanol is gone from DNA - run a gel check on the gel extracted DNA. If the sample floats out of the well you have ethanol contamination.
  • Elute with hot buffer (70°C) and allow the column to incubate with the hot buffer for 5 minutes prior centrifugation
  • Renature the DNA - If the eluted DNA appears to be half the expected size then it is single stranded. Renature DNA by heating it to 95 degrees for a minute and let it cool slowly to room temperature.
  • Run controls - Digest empty vector cut with a single enzyme, perform the gel extraction, and re-ligate it. A vector cut with one enzyme should re-ligate very easily and provide plenty of colonies on the plate. If it does, then the inability to clone the DNA may be related to some other factor, such as secondary structure of the DNA, repeat sequences causing instability in E.coli, or the DNA cloned codes for a protein that may be toxic in bacteria.
  • Minimize exposure to UV light
  • Trim the gel slice as much as possible

-Ligated cultures grew with no RFP signal - TetR repressor protein is preventing the production of RFP. Overnights were made of this cultures.


Thursday-27/07/17

-Church plasmid has arrived - Erythromycin tests begin Make plates using the Church plasmid - streak using pipette tip or inoculating loop

-Experiment with TetR- Pro.RFP : Plasmid prep, restriction digest and run on a gel to confirm parts ligated into plasmid.

-Lanes from left to right - Marker, TetR-Pro.RFP plasmid cut with EcoR1 and Pst, LuxS (Ellen’s experiment). Results are a bit unusual but it seems that the ligation worked.

-Tests using various concentrations of tetracycline to show an increase of fluorescence when tet is added to the system.

-Streaked erythromycin plasmid backbones onto ampicillin plates (x2)


Friday-28/07/17

-Construct primers for G-blocks

-Repeat Tet experiment with the plate-reader.