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Engineering ZBiotics Part 2: How we did it

The techniques we use to make a genetically engineered probiotic


In Part 1 of this series, I discuss what we do when we genetically engineer a probiotic bacteria. In this post, I will explain how we do that.

What I did was change the internal blueprints in a probiotic bacteria. But you may be wondering how I made those changes. This is a very important question, because how a bioengineer changes genetic code can affect the safety or quality of the engineered bacteria. We want to make very specific alterations, while at the same time ensuring we don’t change anything unintentionally.


Homologous recombination

There are several genetic engineering techniques available with the ability to make highly controlled and specific changes. One common technique – the one we use at ZBiotics – is called homologous recombination

Homologous recombination is a natural process that bacteria have evolved over billions of years. It basically functions analogously to a “find-replace” tool in a word processing document. If an external piece of DNA has a stretch of code identical to the bacteria’s own DNA, that identical stretch works as a “find” tool. The bacteria matches the two identical stretches, and then it “replaces” the DNA fragment next to that identical stretch in their own DNA with the DNA fragment next to that identical stretch in the external DNA. (for more explanation see the beginning of this video 0:35-1:10). 

Essentially, we’re taking our new desired genetic blueprint (the piece of DNA with the new instructions we want to give the bacteria), and swapping it in for the bacteria’s original genetic blueprint – the one we want to replace – by flanking our new instructions with matching identical stretches of DNA on either side.

It works like this. First, we identify the spot in the bacteria’s genetic code where we want to insert our new genetic blueprint. Then we read the DNA stretches on either side of that spot, and, with that sequence in hand, design new fragments of external DNA that perfectly match them. Then we connect these matching fragments to each end of the new genetic blueprint we want the bacteria to take up.

Now, we mix this new DNA together with the bacteria. Incredibly, the bacteria will recognize the identical stretches of DNA we created and, using its own machinery, naturally swap in the blueprint we designed between these stretches, just as its evolved to do over millennia. In other words, the bacteria recognize homologous (identical) DNA and recombine (replace) it in their own genetic code; thus homologous recombination.



Building and error-checking

To build the first ZBiotic – B. subtilis ZB183 – I took the blueprint for the anti-acetaldehyde protein. Then I sandwiched it between DNA that was identical to the DNA flanking the blueprint for the flagellin protein I wanted to replace. The bacteria then recognized the identical code and swapped in my blueprint for the unwanted flagellin blueprint. After the swap, I had a new B. subtilis bacteria, complete with my rewritten genetic code.

We’re not done yet, though. We have to make sure that we make as few changes as possible and can track exactly what we do. We don’t want to introduce unexpected additional changes, and we certainly don’t want to introduce any dangerous traits like antibiotic resistance.

The good news is that with recent huge advances in DNA sequencing technology, we can now cost-effectively read the entire genome of a bacteria. That means we can verify exactly what we’ve done and – importantly – confirm that we haven’t done anything else unexpected. So for a few hundred dollars, I got the entire genome of my new bacteria sequenced and was able to verify that everything was correct. 

I then repeated the same process to edit the “off” switches, and bing-bang-bong: I precisely engineered a predictably useful microbe!


A unique, custom-built probiotic

So what do we have at the end of all this? A safe probiotic bacteria, just like what we started with. It behaves exactly as it did before, but with one specific change. It no longer occasionally makes flagellin protein. Instead, it continuously makes anti-acetaldehyde protein. 

That change was specific, highly controlled, and known to be completely safe. We merely took a bit of genetic code that we already knew was safe and added it to a bacteria we also knew was safe. We then went back and checked every single bit of genetic code across the bacteria’s whole genome to make sure we understood exactly what changed and to verify nothing unexpected happened.

And again, that’s it. That’s genetic engineering!

Sounds simple? Thanks to modern technology, it honestly kinda is! And we can use this same strategy to engineer our awesome friend B. subtilis not just to make proteins that break down acetaldehyde, but also to make proteins with all kinds of other functions!

We’re already working hard to do so. The future is exciting, and while we have our own ideas, we’d love to hear what proteins you’d find useful. What would you build? Let us know, and maybe ZBiotics will make it next!

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