Synthetic biology is seeing rapid advances, but the medical applications have thus far remained largely elusive. But now researchers from the Wyss Institute for Biologically Inspired Engineering at Harvard University and Harvard Medical School (HMS) have developed a tool that can track specific populations of bacteria in the gut of living organisms and document population changes over time.
The researchers used an oscillating gene circuit, known as a repressilator, as a sort of genetic clock to measure growth in a given bacterial population over days in the gut of a mouse. Upon excretion, a sample was then cultivated in a petri dish and formed fluorescent rings to enable the identification of the specific bacteria and its growth pattern in the gut. The research was published in the Oct. 11, 2019, issue of Nature Communications.
"Our goal in our group is really to find real-world applications for synthetic biology circuits. Up until this point, this circuit was cool, but what is it good for? Think of it as a timer; like all oscillators, it's essentially a timer," Pamela Silver, a core faculty member at Wyss and the Adams professor of biochemistry and systems biology at HMS, explained to BioWorld.
She continued, "Because of our interest in the mammalian gut and the dynamic of cells in the gut, which we've done a lot on, we decided to see if we could take this relatively complex circuit and put it into bacteria that then go into the gut and see, one, will it even still work and, two, could it inform us on anything about what goes on in the gut with a single microbe?"
Prior research demonstrated that the circuit would work ex vivo, but that was the first in vivo application. The repressilator comprises three bacterial genes that code for three kinds of proteins: Tn10 TetR, bacteriophage lambda CI and E. coli LacI. Those each block the expression of one of the other proteins, creating a negative feedback loop and causing the protein that it had been repressing to be expressed and block the third protein. That process then repeats cyclically.
Those genes are inserted into a plasmid and then introduced into bacteria. The number of negative feedback loop cycles records how many cell divisions the bacteria have undergone; the repressor proteins are diluted as the bacteria divide so their concentration falls. The cycle repeats after 15.5 bacterial generations, thereby creating an objective measurement.
"Our repressilator is like a watch that always moves at the same speed, so no matter how many different people are wearing one, they will all give a consistent measurement of time. This quality allows us to more precisely study the behavior of bacteria in the gut," explained first author David Riglar, a former postdoc at the Wyss Institute and HMS who now leads a research group as a Sir Henry Dale Fellow at Imperial College London.
Each of the three repressor proteins is paired with a differently colored fluorescent molecule and used to create an imaging workflow called RINGS (Repressilator-based Inference of Growth at Single-cell level) to visually express different time points during bacterial growth.
"As a bacterial colony grows outwards, the repressilator circuit creates these different fluorescent, tree-ring-like signatures based on which repressor protein was active in the single bacterium that started the colony," continued Riglar. "The pattern of the fluorescent rings records how many repressilator cycles have occurred since growth began, and we can analyze that pattern to study how growth rates vary between different bacteria and in different environments."
Snapshot or movie?
The current standard is to use sequencing to get a one-time snapshot – and then to compare those over time to get some information on change in a bacterial population. But this new method offers a means to capture the behavior of a single type of cell population in the gut over several days. Diagnostic applications could include assessing gut inflammation or if there's been exposure to a specific nutrient or drug.
Silver said she is confident in the safety of eventually using genetically engineered bacteria in humans for medical purposes. Wyss also has a therapeutic program on that front to produce drug on demand in the gut. But she noted that there's also an environmental risk of releasing those genetically modified organisms; one approach is to build in a kill switch so those GMOs die once excreted, while another is to create an organism that is unable to exchange genetic information with its environment.
The ultimate ideal for microbiome analytics would be to enable the visual tracking of a full range of relevant bacterial populations in the human gut. But the tool, which was tested here only works with specific kinds of gut bacteria so far.
"That would be the dream. In our paper, we actually showed that the repressilator functions in E. coli, including Nissle, which is the most commonly used human probiotic E. coli. We also showed it functions in Salmonella, which is not too surprising because Salmonella is actually closely related to E. coli," said Silver.
"Ideally, we would like to have it report on all gut bacteria," she continued. "The trouble is you have to build a whole new kind of circuit; the principles of the circuit would be the same, but the details would have to be different because the way those bacteria carry out transcription is enough different from how E. coli does it that you can't simply transport it in. So, somebody would have to go and build new circuits for those bacteria. The dream would be that every different kind of bacteria has its own circuit – and then I have this rainbow in my poop."
The repressilator circuit was given orally to mice, where it remained active for up to 16 days after introduction. The fecal samples were cultivated in a petri dish and then visually analyzed. The RINGS analysis offered successful detection of changes in bacterial growth patterns that were achieved at different stages, such as synchronization of the mice using antibiotics.
The researchers also tested varying bacterial growth rates due to gut inflammation. After being given an inflammation-inducing compound, the mice showed greater inconsistencies in bacterial growth that are associated with imbalances in the gut microbiome.
"This repressilator allows us to really probe the intricacies of bacterial behavior in the living gut, not only in both healthy and diseased states, but also spatially and temporally," Silver said. "The fact that we can re-synchronize the repressilator when it's already in the gut, as well as maintain it without the need to administer selective antibiotics, also means that we can study the microbiome in a more natural state with minimal disruption."