Rice University synthetic biologists have hacked bacterial sensation with a plug-and-play system that can be used to mix and match tens of thousands of sensory inputs and genetic outputs. The technology has extensive implications for medical diagnostics, the study of lethal pathogens, environmental monitoring and more.
In a project spanning nearly six years, Rice bioengineer Jeff Tabor and colleagues conducted thousands of experiments to show that they could systematically switch two-component systems. The genetic circuits use bacteria to feel the environment and listen to their neighbors. Their work is evident from a study published this week in Nature Chemical Biology .
Tabor's group fed the exits from known bacterial sensors and also moved sensors between distance-related bacteria. Most importantly, they showed that they could identify the function of an unknown sensor.
"Based on genomic analyzes, we know that there are at least 25,000 two-component systems in bacteria," says Tabor, professor of bioengineering at Rice's Brown School Engineering and leading researcher on the project. "But for about 99% of them, we have no idea what they feel or what genes they activate in response."
The importance of a new tool that unlocks two-component systems is underlined by the 2018 discovery of two strains of a lethal, multi-resistant bacterium that uses an unknown two-component system to avoid colistin, a last resort antibiotic. But Tabor said that the possible uses of the tool extend beyond medicine.
"This is nature's greatest tax source of biosensors," he said. "Based on the exquisite specificity and sensitivity of some of the two-component systems we understand, it is widely believed that bacterial sensors will surpass what humans can do with today's best technology."
Tabor said it is because bacterial sensors have become females
"Bacteria do not have as sophisticated as eyes, ears or nose, but they travel between very different environments – like a leaf or bowel or soil – and their Survival depends on their ability to feel and adjust to those changes, "he said." "Two-component systems are how they do it," said Tabor. "These are the systems they use to" see "light," smell " the chemicals around them and "hear" the latest community news, which comes in the form of biochemical tweets transmitted by their neighbors. "Bacteria are the most abundant form of life and the two-component systems have been found in virtually every bacterial genome that has been sequenced. most species have about two dozen of sensors and some have several hundreds.
There are more than half a dozen wide categories of two-component systems, but all work in a similar way. SK) component that "listens" for a signal from the outside world and at "hearing" it initiates a process called phosphorylation. It activates the second component, a response controller (RR) that acts on a specific gene that puts it on or off as a switch or up or down as a steering wheel.
While the genetic code for the components can be easily seen on a genomic scan, the dual mystery makes it almost impossible for biologists to determine what a two-component system does.
"If you do not know the signal that it feels and you do not know the gene on which it is acting, it is really difficult," said Tabor. "We know either the input or output of about 1% of the two-component systems, and we both know The researchers know that SKs are typically transmembrane proteins, with a sense domain, a kind of biochemical antenna that points through the bacterial objective outer membrane, each sensor domain being designed to lock to a specific signal molecule or Each SK has its own target ligand and binding with the ligand is what starts the chain reaction which puts a gene on, off, up or down.
Importantly, each two-component system is optimized for a specific ligand, their SK and RR components Similarly, Tabor and studying co-author Sebastian Schmidl at the end of 2013 decided to try to change the DNA-binding domain, the part of the response regulator that recognizes DNA and activates the target gene of the path.
"If you look at previous structural studies, the DNA binding domain often looks like cargo that just hitches a turn from the phosphorylation domain," Tabor said. "Because of this, we believed that DNA binding domains could act as interchangeable modules or Lego blocks. "
To test the idea, Schmidl, when a DFG post-doctor in Tabor's group, returned the components of two light sensors, Tabor's law, had previously been developed, one that responded to red light and others who responded to green. The input of the red light sensor to the output of the green light sensor at 39 different locations between phosphorylation and DNA-binding domains. To see if any of the 39 joints worked, he stimulated them with red light and looked for a green light response.
"Ten of them worked at the first attempt and it was an optimal, specific site where the cormorant seemed to work really well, "said Tabor.
In fact, the test worked so well He and Schmidl thought that they had simply succeeded and split up two unusually well-matched roads. So, the repeated test, first attaching four additional DNA-binding domains to the same response regulator and later attaching five DNA-binding domains to the same sensor pathway. Most of these rewirings also worked, indicating that the approach was much more modular than previously published.
Schmidl, now a biology student at Texas A & M University Systems RELLIS campus in Bryan, left Rice 2016. Co-leading author Felix Ekness, a doctoral student in Rice Systems, Synthetic and Physical Biology (SSPB) program, then up the project, constructed dozens of new chimeras and carried out hundreds more experiments to show that the method could be used to mix and match DNA-binding domains between different bacterial species and between different families of two-component systems.
Tabor knew that a top flight record would require a demonstration of how the technology could be used and detecting the function of a completely new two-component system was the ultimate test. For this, postdoc Kristina Daeffler and SSPB Ph.D. student Kathryn Brink transplanted seven different unknown two-component systems from the bacterium Shewanella oneidensis to E. coli. They constructed a new E. coli strain for each unknown sensor and used DNA binding domain switching to link all their activities to the expression of green fluorescent protein.
While they didn't know the entrance to any of the seven, they knew that S. oneidensis was discovered in a lake in upstate New York. Based on that, they chose 117 different chemicals that S. oneidensis could benefit from sensing. Since each chemical must be tested one by one with each mutant and a control group, Brink must perform and replicate nearly 1000 separate experiments. The work paid off when she discovered that one of the sensors discovered changes in pH.
A genomic search for the newly identified sensor underlined the importance of having a tool to unlock two-component systems: the pH sensor came up in several bacteria, including the pathogen that causes the bubonic plague.
"This illustrates how unlocking the two-component system mechanism could help us better understand and hopefully treat diseases better," Tabor said.
Where does Tabor take that technology next?
He uses it to my genomes of human intestinal bacteria for new disease sensors, including inflammatory bowel disease and cancer, with the goal of constructing a new generation of smart probiotics that can diagnose and treat these diseases.
Technique for fine-tuning two-component biocompensators
Rewiring bacterial two-component systems by modular DNA binding domain replacement, Nature Chemical Biology (2019). DOI: 10,1038 / s41589-019-0286-6, https://www.nature.com/articles/s41589-019-0286-6
Synthetic biologists hack bacterial sensors (2019, May 20)
May 21, 2019
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