Take a typical fish out of water and it won't live long. It gets the oxygen it needs from the water it swims in.

In a similar way, scientists are exploring dependency as a method of controlling what microbes can do and where they can do it.

Microbes - microscopic, single-cell organisms that include such things as bacteria, fungi, algae and viruses, to name a few - already do a lot. Life as we know it - our lives included - would not exist long without the many jobs microbes do within us and all around us. Many are great allies to us. A small percentage can take us out.

Finding ways to harness and steer their many properties and abilities is the focus of much research in synthetic biology, the emerging discipline that takes pieces of DNA - the genetic code - and reconfigures it in new ways.

Aditya Kunjapur, the Thomas Willing Early Career Associate Professor of Chemistry and Biomolecular Engineering at the University of Delaware, has extensive expertise in this work, with interest in expanding the genetic code and a special focus on biosecurity and biocontainment.

In a publication in Nature Microbiology, Kunjapur and members of his lab showed how they have been able to make one microbe dependent on another - a dependence that could one day be used to restrict microbial activity to a specific area.

This is of great significance as new technologies emerging in synthetic biology could help us address a wide array of problems in health, agriculture and the environment. It is essential that these developments come with appropriate and effective safeguards to prevent harm and avoid unintended consequences.

These are challenges Kunjapur and his lab are addressing. They're looking for ways to make better - and safer - use of engineered microbes by ensuring their survival as they pursue their mission and by establishing boundaries to keep them from going off course.

"This could be useful in an environment where you want to ensure that one microbe can survive for an indefinite time, but only in a defined region," Kunjapur said.

In an award-winning essay published in the journal Science in 2024, Kunjapur explained the questions his team is addressing, work it hopes will lead to better vaccines, better immune responses and effective solutions for the increasing problem of antimicrobial resistance that threatens public health around the world.

"Our primary hypothesis is that engineering cells to access a broader chemical repertoire of building blocks can improve live bacterial vaccine efficacy," he wrote.

It's a complex endeavor indeed. The goal is to develop a self-contained microbial system that will deliver special residues to disease-creating cells and trigger stronger immune responses. To enable these next-generation vaccines, Kunjapur and postdoctoral researcher Neil Butler co-founded Nitro Biosciences in 2023.

Now Kunjapur's team has shown one way to limit the survival of a microbe. They trained two strains of Escherichia coli (better known as E. coli) bacteria to work together. One strain was trained to create a non-standard amino acid, an amino acid that is scarce in nature. The other strain was engineered to be dependent on that synthetic amino acid.

Earlier reports in the field had shown that a microbe could be engineered to depend on a synthetic amino acid if that synthetic amino acid was directly supplied.

But the need to directly supply a synthetic amino acid would probably not work well in many contexts. Then-doctoral student Mandy Forti and Kunjapur wondered if they could create a steady supply of the synthetic amino acid by having another microbe produce it, thereby creating a sort of self-contained system. They then showed that the dependent strain was sustained by the synthetic amino acid, with almost no escaping microbes.

"Our whole exploration is how to mitigate that risk," Kunjapur said. "We do that in a laboratory setting. We're not trying to rush to put this into the environment. We're studying the mechanisms to see how the system could break. There is a lot of failure analysis."

Building and testing a self-contained, dependent system was a complex process for Kunjapur's team and involved several significant landmarks:

• First, a strain of E. coli bacteria was trained to make a custom, non-standard amino acid that could sustain another bacterial strain without requiring an external supply.

• Next, a separate strain was trained to use the custom non-standard amino acid to produce a fluorescent protein to show that it could successfully use that custom amino acid.

• Next, a strain was designed that relied on that custom amino acid for sustenance.

• Finally, the two strains were grown together. The dependent strain survived as long as the producer strain was in its environment. It could not survive without that producer.

The dependence of one microbe on only one other specific microbe also worked in a context that included other microbes.

"That was the surprise element," Kunjapur said. "The expectation was that other microbes would ruin biocontainment - that it was a matter of how they would do that, not if they would. But Mandy's system worked. We have created an ecology that in this context looks very exclusive."

Forti, lead author of the paper who earned her doctorate last year, said it was amazing to see how the bacterial strains survived and proliferated and to see that after two weeks there was very little escape.

There are still many questions to answer before this technology would be ready for use outside of a laboratory setting.

"There are so many other variables we can test," Forti said. "We need to look at every possible variable these strains could encounter in the environment."

In addition to Forti and Kunjapur, co-authors included postdoctoral researchers Neil Butler and Michaela Jones and UD senior Defne Elbeyli.