Team:UCC Ireland/Project/Notebook/21/08

Monday-21/08/17

• Made overnights in M9 and LB of AmilCP, Church plasmid, and in m9 controls (Cells only, promoter J23119 plasmid, and Yensi’s GFP inducible control).
• Poured Tetracycline plates and completed making new M9 media
• Decided on a protocol for Tuesday’s experiments and laid out hood

Tuesday-22/08/17

Experiment to measure the Church GFP fluorescence vs the Erythromycin concentration

• Measured the OD’s of the overnights. The OD’s were as follows:
• Refreshed the cells in new M9 media and adjusted their OD’s to 0.05. Placed in a 50mL tube and left shaking for 3hrs.
• After 3 hrs the OD’s were as follows:
• Got a clear bottomed dark plate and pipetted the respective cells into each well. Added the correct concentration of Erythromycin to each well.

Fig. 1. Graph showing average fluorescence of Negative control (Cam resistant bacteria expressing promoter), 0.04µg/mL Erythromycin (Legal limit concentration) and 40µg/mL Erythromycin (Highest concentration used. Average fluorescence is average fluorescence over three replicates minus blank.

Fig. 2. Graph showing all average results. As seen, IPTG induced GFP strain is unsuitable as a positive control due to expression levels far above that of other strains used, drowning out their signals.

Fig. 3. Graph showing fluorescence response to all Erythromycin concentrations minus the positive control.

Experiment was carried out with a lid on the 96 well plate to prevent evaporation of the samples. It was found afterwards that the lid is a significant source of autofluorescence. As seen from the graphs, the negative control has a higher RFU than all of the samples excluding the highest concentration of Erythromycin used (40µg/mL). This was possibly due to the lid.

Wednesday-23/08/17

• Refreshed overnights (AmilCP M9 1%, Cells only, promoter J23119) and made to an OD of 0.05.
• Began AmilCP test on the plate reader. This test will examine the rate of AmilCP production vs the OD of the cells.
• Discovered that the lid of the plate reader fluoresces in the plate reader.. This causes an error in our GFP results.
• Made overnights of TetR and RFP. These constructs will be used to make a new ligated TetR-RFP construct to use as our tetracycline biosensor.

Thursday-24/08/17

• Ran a gel on the pure resuspended gblocks to confirm that there is no contamination between gblocks.

  It seems like there is some sort of contamination in the pure gblocks or the primers are binding and amplifying other sites??

  A gel extraction may have to be carried out to get the correct gblocks.

• 3A Assembly of TetR-RFP plasmid:
  • Plasmid prep of the TetR and RFP respectively. Measured conc. And purity on nanodrop.
  • Restriction digest of the fragments using the 3A restriction enzymes
  • Ran the digest on a gel to confirm the digest worked:
  • Lanes: Ladder, cut TetR, uncut TetR, cut RFP, uncut RFP, cut backbone, uncut backbone.
  • 3A assembly ligation run for 50 minutes with T4 Ligase enzyme.
  • Transformed cells with the new ligated TetR-RFP plasmid.

Friday-25/08/17