Project Description

Treatment improved enormously with ready-to-use therapeutic foods: energy-dense pastes needing no fridge, no clean water and no preparation. They work when the child eats them.

Many children with severe acute malnutrition will not. Appetite loss is so central that it is built into the clinical pathway.

Appetite is the gate. We are aiming at the children who fail that test.

When food reaches the small intestine, its cells make oleoylethanolamide (OEA), a lipid signal that reduces food intake. Levels rise with feeding and fall with fasting.

OEA does not persist. Fatty acid amide hydrolase (FAAH), a serine hydrolase, breaks it down. How much satiety signal exists at any moment is a balance between the two rates.

More FAAH means less OEA, a weaker signal, and a child more willing to eat.

A drug needs purification, manufacturing, often a cold chain, and a correct dose given on schedule. Each of those fails most often where malnutrition is most common.

OEA is made in the small intestine and acts there. A gut organism is already in the right place.

Our chassis is E. coli Nissle 1917, and our enzyme comes from Faecalibacterium prausnitzii, one of the most abundant bacteria in a healthy gut.

Why EcN

• Already a probiotic — sold and licensed for over a century
• Survives the gut — passes gastric transit and colonises
• Non-pathogenic — Risk Group 1, handled at BSL-1
• Engineerable — standard cloning methods work

We cannot engineer F. prausnitzii. It is a strict anaerobe, difficult to culture, and has no established genetic toolkit.

OEA sits in the gut lumen, outside the bacterium. An enzyme made in the cytoplasm is separated from its substrate by membranes it cannot cross.

Rather than guess, we built a panel that tests the question directly. Every variant is the same enzyme with the same 6xHis tag, differing only in where it is sent.

Six of these have designed primers; the catalytic mutant does not, which is why it appears as Not built on the Results page. Because backbone and insert amplify in separate reactions, six constructs meant twelve PCR reactions. Both numbers appear in our notebook and they describe different things.

The last two are controls, and they are why the panel is interpretable. The GFP reporter confirms inducible expression in the backbone independently of whether the enzyme works. The catalytic mutant separates FAAH activity from anything the host does.

We use IVA cloning, which lets E. coli join DNA fragments that share overlapping ends. Backbone and insert are amplified separately, the template is destroyed with DpnI, and both fragments are co-transformed. The cell does the joining.

No restriction enzymes, no ligase, no assembly mix. A new construct costs two primers, which is what let us iterate on a student budget.

Our backbone, pEM106.2, supplies an inducible promoter, chloramphenicol selection and a secretion carrier already in place. Clones are screened by restriction digest and confirmed by Sanger sequencing.

The figure-by-figure evidence is on our Results page, the dated record on the Notebook page, and the assay designed to test the central claim on the Measurement page.