A gene created by researchers at Rice University offers scientists valuable data related to microbes through discharge of gas from the soil. The most recent version is a powerful two-stage microbial sensor that will assist geobiologists, bioengineers and other scientists to monitor gene expression as well as the bioavailability of nutrients in laboratory reproduction of environments such as sediments and soil without perturbing them.
The gas is generated by using microbes genetically engineered to give an account of their environment as well as activity and combined into soil samples in restrained laboratory experiments. A gas that oozes out informs scientists about the number of target microbes that exist, and another gas informs the activities of the microbes. Ultimately, the Rice researchers will expect the programmed microbes to disclose whether and how they communicate with one another.
The sensor has been described in the ACS Synthetic Biology journal published by the American Chemical Society.
The study in progress started in 2015 with the help of a grant of $1 million from the W.M. Keck Foundation and has been headed by Jonathan Silberg, a Rice synthetic biologist; Caroline Masiello, a biogeochemist; and Hsiao-Ying (Shelly) Cheng, a graduate student and lead author of the study. Their aim is to evaluate bioactivity in opaque environments, specifically those in which modifying the environment will change the outcomes.
According to Silberg, the new gas-emitting microbes function on the same principle that governs those that include two fluorescent proteins; for instance, a green-fluorescing protein will tag all the cells in a dish, and a red protein will get illuminated when triggered by microbial activity, such as proximity of a specific molecule or protein expression.
“In those systems, you can check the ratio of green to red and know, on average, what the cells are doing,” he stated. “But that doesn’t work in soils.”
At present, scientists evaluate microbial activity in soil by crushing samples and adopting processes such as high-performance liquid chromatography to quantify their constituents. This removes the chances of analyzing the same sample over time, and also restricts the scope of the data.
“Our system answers the right question,” stated Masiello. “Do microbes know these compounds are present, and what are they doing in response to them?”
In the ratio-metric system developed at Rice lab, gases discharged from modified Escherichia coli or other microbes can assist researchers in evaluating soil development. The term ratio-metric indicates that the gas output is directly proportional to the input, which is the level of activity sensed by the microbe here.
In one of the tests, E. coli was transformed to expel enzymes that produce bromomethane and ethylene. The microbe continuously produced ethylene, thereby enabling the researchers to observe the microbe population size; however, it produced only bromomethane when triggered by, here, bioavailability of acylhomoserine lactones (AHL), molecules enabling signaling between bacteria.
Once Cheng placed the E. coli in agricultural soil and fixed the temperature to increase gas signals, she discovered that the addition of short- and long-chain AHL did not have an impact on ethylene output but drastically impacted bromomethane. The highest concentration of short-chain AHL elevated the bromomethane signal by over an order of magnitude, and that of long-chain AHL elevated it by nearly two orders of magnitude.
Investigations with a different bacterium, Shewanella, with sediment as a native habitat, revealed similarly robust outcomes.
“The dynamic range for sensing chemicals with what Shelly’s built is very good,” stated Silberg. “It will vary with the organism, but synthetic biology is really about tuning all of that.”
The particularly useful aspect of this work is the potential to distinguish between what’s chemically extractable in a marine or soil environment and what a microbe perceives is there. Just because we can grind up a soil and measure something doesn’t mean that plants or microbes know what’s there. These tools are what we need to be able to, for the first time, measure microbial perception of their environment.
Caroline Masiello, Biogeochemist
The transformed microbes are meant to be applied for lab investigation, as opposite to in the open. But tests would be much faster than current processes and allow labs to monitor a sample continuously over time. The researchers anticipate applications not only in synthetic biology and environmental science but also for tracking the environmental fate of gut bacteria being developed for diagnostics and therapeutics.
In the future, the Rice lab aims to focus its attempts on the conditional output portion of the sensor.
As we’ve been building this, people like (Rice bioscientist) Jeff Tabor and others are standardizing the sensing modules. We’re building new output modules that you could then couple to the great diversity of sensors they are building.
Shelly’s really led the way to prove that we can do gas reporting, and she was the first to do it in soils. She then showed we could do it with horizontal gene transfer as part of our proof of concept, and now this. The tools are just getting there, and I think applications will be next.
Jonathan Silberg, Rice Synthetic Biologist
Graduate student Ilenne Del Valle in the Systems, Synthetic and Physical Biology graduate program, research scientist Xiaodong Gao, and George Bennett, the E. Dell Butcher Professor of Biochemistry and Cell Biology, all from Rice University, are the co-authors of the paper. Silberg is an associate professor of biochemistry and cell biology. Masiello is a professor of Earth, environmental and planetary sciences.
The W.M. Keck Foundation, Rice University, a Taiwan Ministry of Education Scholarship, the National Science Foundation Long-term Ecological Research Program, Michigan State University AgBioResearch, and the Department of Energy, Offices of Science and Energy Efficiency and Renewable Energy supported the study.