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New ways to control bacterial factories for future biotech uses

The lab of Cheryl Kerfeld has developed a new method to manipulate miniature factories found in bacteria that could someday lead to new medical, industrial, or energy applications.

The factories, called expand iconbacterial microcompartments – or BMCs – are found in bacteria all over the world. They are very flexible in variety and function, which is why scientists want to create synthetic versions, modeled on the real thing, to perform new functions that benefit human beings.

But the factories can be tough to work with in the lab.

With the new method, scientists can build factories in test tubes, allowing for high levels of control. Then, they change the electric charge on the inside of the factory walls, or shells, and attract desired cargo inside them, resulting in custom factories with new uses. The study is published in ACS Nano Letters.

Putting factories together on demand

BMC functions vary, depending on the host, which could be a photosynthetic bacterium in the Arctic or a pathogenic bacterium in your gut. But their outer walls are made of the same building blocks. Basically, these are three types of expand iconprotein tiles that snap together to form a shape like a soccer ball.

“We want to control how and when these building blocks assemble into a wall. However, on their own, some of them assemble in unproductive ways. That dynamic makes it impossible to isolate and work with them,” says Andrew Hagen, a post-doc in the Kerfeld lab.

GIF of protecting group
The new technique controls factory assembly, on demand
By Andrew Hagen, Igor Houwat, MSU-DOE Plant Research Laboratory, 2018

So, the team created a way to thwart factory assembly and then trigger it on command. They genetically fused an additional protein domain that functions as a “protecting group,” to one of the protein building blocks (BMC-H) of the factory. This fusion prevents the pieces from coming together and forming these microcompartments.

After all necessary factory components are added, the scientists add an expand iconenzyme that cuts off the protecting group. Then, the proteins can snap together to make the factory walls. The effect leaves no scars or remnants of the protecting group.

“This level of control will help us to isolate the proteins, manipulate them, study them, make them shelf stable,” Andrew says.

Proof of concept

The team then tried to incorporate inorganic molecules inside the factory walls, using the new method. But they had to come up with a couple more tricks.

Jeff Plegaria and Bryan Ferlez, both Kerfeld lab post-docs, switched two negatively-charged expand iconamino acids inside the BMC-H protein tile into positively-charged ones.

Then, they introduced negatively-charged cargo to the mix. In principle, the opposing charges would attract cargo to the building blocks, causing them to attach to each other.

GIF of the new encapsulation method
A new method to introduce cargo into synthetic factories
By Andrew Hagen, Igor Houwat, MSU-DOE Plant Research Laboratory, 2018

“We tried incorporating both inorganic, negatively-charged gold nanoparticles, and a fluorescent protein fused with an extra negatively charged piece,” says Jeff. “The result was successful. Our microscopes showed both types of cargo adhering to the inside of factory walls once those snapped into formation.”

In the case of the fluorescent protein, the negative charge was the Velcro that glued the cargo to the wall. In theory, one could add this Velcro to their favorite protein that they want to target into the factory.

“This proof of concept of building factories in test tubes holds exciting promise for the field,” says Bryan. “We are showcasing the ability to put a whole new type of molecular machinery inside the factories. And these developments help us look toward the future of applying this technology.”

For example, some medical imaging technologies rely on inorganic materials like the gold nanoparticles. The new method could eventually use repurposed BMCs to safely ferry such cargo around the body for imaging purposes.

Beyond carrying exotic new materials, the factories will also be easier to study by researchers trying to understand the basic science behind their assembly. Previous methods try to ‘graft,’ grow, and study the factories inside other living bacteria. However, so much goes on in those bacterial systems that gets in the way of studying the factories.

“Now, we can study factory wall assembly in a test tube, where we can use analytical methods that are impossible to do in a living cell,” says Andrew. “We also have evidence the method works with factories from diverse bacterial species. That means researchers could apply it to their particular bacterial microcompartment of interest. They will also be able to more rapidly build prototypes of custom factories.”

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