IGEM:IMPERIAL/2006/LabCalendar/2006-9-6

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TO DO:
*Maxi & mini of J37036 & J37018
*Send J37036 for sequencing
*Make frozen stock of new T9002
*Culture J37022 for maxi and mini and freezing & TESTING on Thurdsday
*Culture 1st step of ligation for final predator with new aiiA for maxi & mini on Thursday

Maxiprep

  • 4 x J37036 (a,b,c,d)
  • 2 x J37018 (a,b)

Miniprep of J37018 & J37036

Testing of J37015

  • 2mL overnight culture was grown

In order to test for protein expression (using the GFP attached to the gene):

  • Diluted into OD 0.1 (8mL) at 10:40
  • Dilution again into OD 0.1 at 12:10 (OD was at 0.688)
  • Taken out of shaker at 15:00 - dilute again into OD 0.1
  • Inoculate diluted culture with 3 different AHL concentrations:
  1. 10nM AHL
  2. 1nM AHL
  3. 0.1nM AHL
  4. 0nM AHL (Control)
  • Put into shaker at 16:00 - leave in shaker to allow GFP expression, kicking off the positive feedback loop with initial AHL concentrations from above

In order to assess any AHL levels that have accumulated overnight:

  • Took morning culture of J37015 (about 1mL left) and spin down the cells to get the supernatant
  • J37016 is diluted to OD 0.1 and 1200μL is inoculated with 800μL of supernatant of J37015 of morning culture
  • Put in shaker at 15:15 - to be taken out at 19:00 (leave for 4hrs for GFP expression to reach steady state)
  • Both above experiments taken out at 19:00 & read with Wallac Victor III

Activity of Acylase

  • Made up enzyme/AHL mix using Enzyme Activity Protocol, row H
  • Took 2mL of diluted J37016 culture
  • Spin down the cells
  • Remove supernatant
  • Add 2mL of enzyme/AHL mix
  • for control: 1980μL of J37016 & 20μL AHL (100nM final concentration)
  • Put both 5mL tubes into shaker at 16:25
  • Taken out at 19:00 & read with Wallac Victor III

Sequencing

  • Sequenced J37036 - using reverse plasmid primer for A2 (Tm = 56[[:Category:{{{1}}}|{{{1}}}]])

Culturing

  • Cultured up 2 colonies in 110ml of J37022 from Plate 1 for maxi and mini
  • Cultured up 2 colonies in 110ml of J37022 from Plate 2 for maxi and mini
  • Cultured up 2 colonies in 110ml of aiiA for maxi and mini
  • Cultured up 2 colonies in 2ml of J37022 from Plate 1 for mini
  • Cultured up 2 colonies in 2ml of J37022 from Plate 2 for mini
  • Cultured up 2 colonies in 2ml of aiiA for mini
  • Cultured up J37016 in 2ml using frozen stock
  • Cultured up J37015 in 2ml using frozen stock

Electroporation of Dr. Fray's plasmid containing aiiA

  • Was not successful because no autoclaved cuvettes were ready to be used
  • Looked for any possibly usable cuvettes in vain
  • This has to be done first thing tomorrow morning (so we might get colonies in the evening)!!!
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