The Michigan State University 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 bacterial 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.

However, 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. 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.

BMC functions vary, depending on the host, which could be a photosynthetic bacterium in the Arctic or a pathogenic bacterium in your gut. Their walls are made of the same building blocks. These outer walls are made of three types of protein tiles that snap together in a shape like a soccer ball. 

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

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

After all necessary factory components are added, the scientists add an enzyme 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 protein. 

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

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 postdoctoral students, switched two negatively-charged amino 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. 

“We tried incorporating both inorganic, negatively-charged gold nanoparticles and a fluorescent protein fused with an extra negatively charged piece,” Plegaria said. “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,” Ferlez said. “We are showcasing the ability to put a whole new type of molecular machinery inside the factories. 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,” Hagen said. “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.”