Difference between revisions of "Team:York/ProjectBreakdown"
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<p>Using the <a href="http://www.abcam.com/phosphate-assay-kit-colorimetric-ab65622.html"> ABCAM </a> 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.</p> | <p>Using the <a href="http://www.abcam.com/phosphate-assay-kit-colorimetric-ab65622.html"> ABCAM </a> 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.</p> | ||
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Revision as of 10:57, 16 September 2015
Project Breakdown
Molecular Biology
- Some info
- More info
Phosphate Assay - Measuring orthophosphate and polyphosphate levels
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.
Growth Assay - Measuring Phil's growth
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.