IGEM:MIT/2006/Notebook/2006-7-6

Stationary phase promoter

 * Plate reader data for first test of stationary phase promoter.

Control Promoter Culture Growth:

http://openwetware.org/wiki/Image:Control_Growth.png

Control Promoter Fluorescence:

http://openwetware.org/wiki/Image:CPF.png

Stationary Phase Promoter Culture Growth:

http://openwetware.org/wiki/Image:Stationary_Growth.png

Stationary Phase Promoter Fluorescence:

http://openwetware.org/wiki/Image:SPFF.png

GC Progress
We finetuned our program for the machine, optimizing retention time. We also found what dilutions of our solution will be ideal for GC measurement (1:10000 dilution of methyl salicylate in n-heptane). We have quantitative data in the form of 2 graphs displaying the proper peaks for two different dilutions of pure methyl salicylate in n-heptane. The procedure we followed was:

^Why we think the max. concentration of methyl salicylate in the original extract is 1&mu;L/mL: we added 40&mu;L of SA to 10mL of culture, the max concentration of methyl salicylate that we could have gotten was around 4&mu;L/mL. But then we took 5mL of the supernatant (so, 20&mu;L of methyl salicylate) and extracted into 20mL of hexane, so the max. concentration in the hexane would then be 1&mu;L/mL.
 * Pellet the cells and add 5mL of supernatant to 20mL of n-heptane in a sep funnel.
 * shake the sep funnel, and then put 1mL of the hexane layer into an eppendorf to take to the GC. The max. concentration of methyl salicylate in this is around 1&mu;L/mL.  When we ran a 1:10 dilution of this, there was lots of column bleeding. we're not sure why, but we think it might have been due to polar compounds.^
 * We made a 1:10 dilution of methyl salicylate to use as a stock, and then diluted this to 1:1000. The 1:1000 nearly burned out the filament, so we then diluted the 1:1000 to 1:10000 and 1:100000. The 1:100000 yielded a very small peak, and the 1:10000 was perfect.