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From 2013.igem.org

Overview

We want a bacteria efficient in metabolize fatty acid and liberate electrons to the media. For this we have THREE approaches:

1° Avoid the FadR (protein that prevents the break down of fatty acid) expression

We know Shewanella doesn’t metabolize fatty acid all the time, mainly due to the media composition. If it doesn’t have fatty acid, the bacteria won’t produce the enzymes that degrade the fatty acid. But if you have fatty acid, then yes, enzymes that break it will be produced. So, to not produce such enzymes, regulatory proteins, such as protein FadR inhibitory enter in scene. FadR is a protein that inhibits the metabolism of fatty acid, she connects on the operator preventing the RNA polymerase to continue and transcribe the information to produce proteins that bacteria need.

How are we going to avoid de FadR expression?

Forward and Reverse primers were constructed for parts of the FadR gene, a 5’ and 3’ pieces were amplified (up and down) in order to design a construction in this following structure: gene up + tetracycline resistance gene + gene down.

So the wild, normal gene leaves and the construction takes place by recombination.

2° Increase the FadL expression.

In Shewanella putrefaciens, there is a protein that works in the intaking fatty acid called FadL. The FadL gene we introduced begin to overexpress this proteins, enabling that more fatty acids enter inside the cell and the generation of ATP and electrons increases.

3° Increase the FadD expression.

The fatty acid enter in the bacteria trough FadL protein, soon, it is gonna be catalized for a serie of proteins on bacteria intern membrane. One of this proteins is the FadD that works ligating CoA in fatty acid chain preparing it to get into the Beta-Oxidation metabolic pathway.  The FadD gene were introduced in a vector and transformated  in S. putrefaciens. She will begin to overexpress these proteins, signaling and enabling that more fatty acids enter inside the cell to be degradated.