Quick note from the Bibel lab 6 - after a freeze thaw, our MDH’s still got it!

Quick note from the Bibel lab 6 - after a freeze thaw, our MDH enzyme’s still got it!

More excitement today as Nicholas and I discovered that the malate dehydrogenase he purified is still super active after a freeze thaw. So active we have to dilute it 150-fold to use in these MDH activity assays.

The below is copy/pasted from my update last Friday for context…

*Basically it shines light through a sample in a cuvette and measures how much light makes it through. (And doesn’t use floppy discs!!!!) It has tons of individual diodes that allow you to measure light absorbance at tons of individual wavelengths). We only cared about one (340 nm), but they wanted to do a larger spectra to demo what the machine could do.

But we were more interested in what our enzyme could do! NADH absorbs 340 nm light, so we could mix our purified mdh, NADH, & oxaloacetate and measure the NADH go bye-bye (get oxidized to NAD+ as oxaloacetate gets reduced to malate). https://www.mdhcures.org/methods-and-...

We didn’t have a chance to really test the enzyme much. But we at least saw it’s really active – in fact, we kept having to dilute it further to get a concentration at which we could actually get usable data (the first time it was over before we could even start collecting). Thankfully we have TONS of this bad boy. Like over 20 mg! From only 400 mL media. MDH can crash at higher concentrations, so we froze all ~15 mL worth of our dialyzed proteins in 30 μL aliquots in PCR strips. It was a lot of tedious transferring to tubes, but now we have sooooooo much enzyme to play with “guilt-free” since we can take just a single aliquot whenever we want to test anything. The rest will still be safe and that single aliquot will literally let us run hundreds of assays!

Background: Our lab investigates how bacteria rewire their metabolism to facilitate the process of “bioremediation” – using living organisms to clean up the environment. We’re most interested in the bioremediation of the toxic heavy metal chromium, Cr(VI). High levels of Cr(VI) are incompatible with most life. However, some bacteria are able to not only survive in its presence, but also reduce it to a much less hazardous form, Cr(III). A sort of biological "detox" if you will. This form of remediation is much more environmentally-friendly and sustainable than conventional chemical and physical methods, but the metabolic adaptations involved are not well-characterized. By unraveling these adaptations in the largely-uncharacterized bacterium Bacillus safensis, we hope to help advance the use of this microbe for environmental clean-up. 

We are interested in how Bacillus safensis is able to metabolically adapt to extreme circumstances, such as during Cr(VI) bioremediation. MDH is at the core of metabolism, and thus a great place to look. Through recombinant protein expression, we got E. coli cells to make a lot of the Bacillus safensis MDH & purified it. And now we’re characterizing its enzymatic activity. Having the pure protein lets us manipulate conditions to test its limits, how it's affected by its environment such as pH, temperature, even the presence of metals. We can even make changes to the protein's sequence to tease apart how it works. In vitro is great for that sorts of things, but not very realistic. Therefore, back at the organismal level, we're comparing the metabolism of different strains and bacterial species in the presence of metals, and doing some genetic engineering, we aim to help tease apart both conserved and unique metabolic adaptations. 

Simultaneously, we are working to put this knowledge to practical use. We are optimizing the bioremediation potential of Bacillus safensis with hopes of using it to clean up heavy metal-contaminated environments and promote the growth of plants in once-barren landscapes. 

I’m so grateful to be working with phenomenal students – Rory, Nicholas, and Haley!

More about the Bibel lab:
* official site: https://sites.google.com/stmarys-ca.e...
* blog: https://thebumblingbiochemist.com/upd...
* YouTube playlist:    • Quick "lab notes" from the Bibel lab  

more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 or search blog: http://thebumblingbiochemist.com