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:
- 10nM AHL
- 1nM AHL
- 0.1nM AHL
- 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)!!!