After proving that two of our ENA fusion constructs gave a consistent, rapid decrease in dissolved calcium concentrations, we wanted to prove that our proteins are to thank for that and add proof to their proposed mode of action as biomineralization catalyst (see design). That’s why we wanted to catch them in the act.

To do so, ENA fusion fibers were added to hard water, recovered by sedimentation, and taken to a transmission electron microscope (better known as TEM) to visually inspect wild type ENA fibers and our ENA fusion constructs. Look at the pictures down below and see our proteins in action! We’ve provided some notes to help you analyze them. One thing to keep in mind: all pictures were taken without addition of negative staining salts such as uranyl acetate. This results in lower contrast images of the fiber features, but allows us to better appreciate a possibly altered contrast by calcium binding,

Let’s start with getting to know ENA a little better;

Now, when we place our water softening ENA fusion fibers in hard water we see the following occur!

Exciting right? Let’s analyze.

Macroscopically it looks like this:

Unlike WT ENA fibers, addition of our active ENA fusions to hard water results in a rapid increase in turbidity, followed by sedimentation of an insoluble aggregate of entangled fibers and associated calcium carbonate crystals. Hurrah! We’re precipitating out calcium carbonate from our water. ​

So what about cost and efficiency?

If you’re more into numbers; the experiment described in depth in the engineering success page might be more enticing to you. There, through a titration that indicates free calcium, we proved that our constructs had a decrease of up to 25% in different commercial water types. Here you can see the final results, with the negative control being the titration results for just the pure water.

The way we think our protein works is by catalysing the reaction from calciumcarbonate in solution to a solid crystalline form. Another way it could be reducing free calcium is by just binding it. Where we don’t have conclusive proof that shows that this isn’t happening, we do have some strong indications.

That last point is shown by this graph. This indicates our protein could work in a setting of heatened water to remove supersaturated calcium carbonate to relevant levels, all tests were done after 1 minute indicating a fast working. In the future we would like to get rid of the heating step by improving amounts of calciumcarbonate formation by for example adding carbonic anhydrases.

Finally, to start getting an idea on costs of implementing our solution the production capacity for E. coli was tested and showed the following results.

Impressive yield, no? We find that up to 54% of the E. coli wet weight consists of our ENA fusions, which are quite conveniently recovered by cell lysis and lysozyme treatment, followed by boiling in SDS and precipitation of the resulting ENA fusion slurry. That’s it, this is our active product, which can be diluted at least down to 290*10^-9 mg / mL whilst retaining full water softening capacity. That means, theoretically, starting from 1L of E. coli culture, we can soften up to 160*10⁹ L of hard water. Leading us to believe this might work on relevant volumes in an industrial setting.