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

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Number of Transformants from 7/5 SAMT-, BAMT-, & ATF1-BB Ligations/Transformations

pUC19 E0040BB (+Ctrl) BSMT-BB (+ Ctrl) BAMT-BB SAMT-BB ATF1-BB
70 lots (~300) 11 1 14 1

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:

  • 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 1μL/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.

^Why we think the max. concentration of methyl salicylate in the original extract is 1μL/mL: we added 40μL of SA to 10mL of culture, the max concentration of methyl salicylate that we could have gotten was around 4μL/mL. But then we took 5mL of the supernatant (so, 20μL of methyl salicylate) and extracted into 20mL of hexane, so the max. concentration in the hexane would then be 1μL/mL.

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