1. Inorganic phosphate ions enter the periplasm through the PhoE phosphorin.
2. Once in the Periplasm phosphate can enter the cytoplasm in one of two ways – non-specific (through Pit) or specific transport. For the latter, the substrate binding protein (PstS) of the Phosphate-specific ABC transporter (Pst) binds the phosphate ion.
3. It then brings the ion in close proximity to the transporter’s transmembrane domains – the permeases PstC and PstA. These two proteins provide the translocation pathway and the phosphate’s uptake is energized by a homodimer of PstB (an ATP-ase protein that hydrolyses ATP).
4. Phosphate ions can then be used for cellular metabolism which includes ATP formation from ADP and phosphate by the F-ATPase protein. ATP is the substrate for the Polyphosphate kinase enzyme (PPK1) which reversibly transfers the γ-phosphate of ATP onto a chain of polyphosphate (PolyP). Amongst many roles polyphosphate acts as energy and phosphate storage molecule.
5. Polyphosphate can be used to generate ATP from ADP and in the case of phosphate shortage, the cell releases orthophosphate residues using the Exopolyphosphatase (PPX) enzyme which cleaves a residue at a time from PolyP’s

In order to determine whether our organism "Phil" was accumulating phosphate, we had to develop a test for quantifying the levels of phosphate in media and within our bacterial cells. We developed a reliable technique to measure both orthophosphate and polyphosphate concentrations in cell supernatant and within growth media.

Using the ABCAM kit (ab65622) containing malachite green and ammonium molybdate as indicator components, we enhanced the existing protocol to improve the reliability and accuracy of our tests. This was necessary following preliminary experiments on E.coli supernatant showing that the unaltered assay did not detect polyphosphate. Building on multiple tests, we incorporated an acid hydrolysis and neutralisation step in order to accurately measure polyphosphate levels.

To be able to tell whether Phil was going to work in environments with variable phosphate concentrations, we devised an assay to measure his growth rates. Using the KEIO collection, the biggest collection of knockout strains of Eschericia coli, we were able to determine the effect of each gene we were tampering with. Knocking out phosphate transporters exhibits a growth phenotype, and based on modelling results we expected a change in the growth phenotype if phosphate accumulates.

Growth assays were based on optical density measurements of cell cultures at 650nm over a set period of time, either 7 or 24 hours. The growth rates and curves of multiple phosphate transporter knockout strains have been compared and analysed in an attempt to find both a chassis for characterisation of genes, as well as experimentally test for a decrease in cell growth, as predicted by computer modelling.

Our experiments showed that PstC knockouts had the biggest change in growth compared to wild-type strains.

Fine Details

What is MOPS media?

Shortened for "3-(N-morpholino)propanesulfonic acid", it has an almost completely neutral pH and can be used as a replacement media for LB that doesn't have phosphate in it. This allowed us to measure growth at variable concentrations of phosphate.