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

 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)

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 1200 is inoculated with 800 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: 1980 of J37016 & 20 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)

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)!!!