If there is a modified E. coli strain and we want to observe if it produces more fatty acids than the non-modified, what kind of analysis, tools, equipment are needed? Is there a simple way?
Thank you for your attention and time.
Dear Dimitrios Eleftheriou ,
I recommend reading a number of papers (from the group) of William Dowhan. He and his co-workers are experts in E. coli mutations that alter lipid (and/or fatty acid) composition, see for example:
Rowlett, V. W., Mallampalli, V. K., Karlstaedt, A., Dowhan, W., Taegtmeyer, H., Margolin, W., & Vitrac, H. (2017). Impact of membrane phospholipid alterations in Escherichia coli on cellular function and bacterial stress adaptation. Journal of bacteriology, 199(13), e00849-16
And
Mishra, N. N., Bayer, A. S., Tran, T. T., Shamoo, Y., Mileykovskaya, E., Dowhan, W., ... & Arias, C. A. (2012). Daptomycin resistance in enterococci is associated with distinct alterations of cell membrane phospholipid content
In essence the lipids are extracted by a ‘classical’ Bligh & Dyer and fatty acid composition is analyzed by Gas-liquid chromatography after conversion to their methyl-ester form (using fatty acid standards).
Best regards.
Rob Keller, one more question; If we just measure the acidity of F.A. produced in the culture media - wouldn't we get an indication of F.F.A. production (controls will be used)? Thank you.
Dear Dimitrios Eleftheriou ,
The point is that stating “a modified E. coli strain that produces more fatty acids than the non-modified” is multi-interpretable since fatty acids are out there in various forms:
-Primarily (I think) built in phospholipids (two fatty acids esterified)
-Built in triglycerides (three fatty acids esterified)
-Built in lysophospholipids (one fatty acid esterified)
-Present as free fatty acids (one fatty acid per FFA)
This leads to the following considerations:
-Consequently, the extraction efficiency (might) varies of the above variations of fatty acid containing substances
-Measuring the amounts requires different methods (acid number of fatty acids is focused on FFA content)
As a crude measure your suggestion might tell you something (just try I would say…), I can imagine that ‘feeding’ the cell culture with a certain amount of FFA and measure how much FFA is left after a certain time might give differences for the two strains (modified and wild type).
Best regards.
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