Team:Aberdeen Scotland/Switch Characterisation


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University of Aberdeen - ayeSwitch - iGEM 2010


Switch Characterisation Lab-Diary

Prep Week (7th – 11th of June)

  • 1.Confirmation of successfully integrated cassettes at the Trp1 locus of:
    • i.N25 with Pgal1 – GFP
    • ii.N4 with Pcup1 – GFP
  • 2.Confirmation of GFP expression of N4 and N25 using microscope.
  • 3.Promoter characterisation: GFP induction experiment using the fluorometer on:
    • i.BY4741: N25 with Pgal1 transformants
    • ii.BY4741: N4 with Pcup1 transformants

Week1 (14th – 18th of June)

  • 1.Transformation of pRS414 and pRS415 into BY4741ΔTrp
  • 2.Quantitative determination of the doubling time of BY4741ΔTrp
  • 3.Yeast (BY4741) transformation of constructs:
    • i.pRS415
    • ii.pRS414

Week2 (21st – 25th of June)

  • 1.Dose response experiment using Pcup (from N4)
  • 2.Research for parameter values for theoticians models
  • 3.Doubling Rate of N25 grown on SD with Raffinose as a carbon source
  • 4.FACS experiment of both Prs414 and prs415, dose response and Time induction set up but experimental and construct errors meant the experiment had to be trouble shooted and then tried again in the future.

Week3 (28th – 2nd July)

  • 1.Further research for parameter values for theoticians models.
  • 2.Problem solving experiment to determine why no fluorescent expression was observed from the previous FACS. Microscope (using fluorescent filter) observations of fluorescence in:
    • i.N4
    • ii.N25
    • iii.pRS414
    • iv.pRS415
  • 3.GFP expression: pRS415 dose response (0.5, 1, 2, 3, 4, 5% of galactose) using the Fluorometer.
  • 4.GFP(pRS415) and CFP(pRS414) expression using FACS analysis
  • 5.Time induction experiment using Pcup (from N4)
  • 6.Transforming pRS414, pRS415 and pMS2 into E.coli (bulking them up)

Week4 (5th – 9th of July)

  • 1.Designing primers to modify pMS2 (replacement for pRS414)
  • 2.Checking the presence of Age1 in pMS2 / Prep of cut pMS2
  • 3.Transformation of pMS2 into BY4742
  • 4.Digest of YCp lac 22 Fl
  • 5.Time induction experiment of Prs415 on the fluorimeter, no valid results obtained due to sample being frozen overnight.

Week5 (12th – 16th of July)

  • 1.Homologous recombination of CFP into YCp lac 22 Fl
  • 2.Microscope analysis of YCP-CFP expression
  • 3.Made Master plates of BY4742 transformants

Week6 (19th – 23rd of July)

  • 1.Time inhibition of GFP expression of pRS415 when inhibitor added on fluorometer.
  • 2.Time induction of GFP expression on fluorometer.

Week7 (26th – 30th of July)

  • 1.PCR amplification of Bbox and CFP (from pRS414) and GFP (from pRS415)
  • 2.Ligated BBox into pMS2 and transformed into E.coli
  • 3.PCR of N-GFP homologous sequences
  • 4.Transformation of BY4741 ΔTrp with pRS414 and N-GFP (homologous recombination)
  • 5.Made master plates of BY4741 pRS414-N-GFP transforms

Week8 (2nd – 7th of August)

  • 1.Experiment observing the effect of MS2 (under control of Met17) on expression of GFP (415) [microscopy + fluorspar readings]
  • 2.Designing primers for amplifying Cup1-2 (from N4)
  • 3.Tested BY4741 pRS414-N-GFP transformants

Week 9 (9th – 14th of August)

  • 1.Encountered problems with growing pRS415 for planned FACS induction experiments, thus replaced plates to replenish nutrients, experiments to be repeated week beginning 16th of August.
  • 2.FACS analysis of MS2 vs GFP expression
  • 3.PCR amplification of CUP promoter form N4 and transformation into prs414
  • 4.PCR Colony screening pRS414-N-GFP transformants