Common lab bacteria have been turned into scavengers that seek and destroy the herbicide atrazine, an environmental pollutant that can be harmful to wildlife. Key to the transformation is the combination of a synthetic switch that allows the bacteria to chase the chemical and a gene taken from another species of bacteria for breaking down atrazine.
Some wild bacteria have evolved the ability to metabolize atrazine. Using a synthetic biology approach, a team at Emory University in Atlanta has now equipped a synthetic strain of E. coli with the ability to hunt down atrazine and metabolize it.
Bacteria normally use sensory proteins called chemoreceptors to spot chemicals in their environment. Reengineering one of these receptors into a designer protein that recognizes atrazine would have been a formidable challenge. So Justin Gallivan and his team instead turned to RNA to develop an atrazine-binding molecule called a riboswitch.
“A riboswitch is a piece of RNA that binds to a small molecule and changes shape when it does that, which then leads to a change in gene expression,” explains Gallivan. His group used a novel selection process to synthesize and evolve a new riboswitch from scratch in the lab. Coupling the riboswitch to a gene that controls movement allows bacteria to move toward nearby atrazine.
The team synthesized a quadrillion (10^15) pieces of RNA, each with a randomly ordered sequence of 40 nucleotides, and tested their ability to stick to atrazine. After repeating this for several rounds, and removing all the RNAs that bound the breakdown product of atrazine, the researchers had selected a much smaller selection of sequences that all stuck to atrazine.
The riboswitch also needs to be able to change shape in such a way that it only allows the protein to move when atrazine is present. Gallivan’s team fused the atrazine-binding sequences to another large selection of random RNA sequences, each a potential candidate for switching shape in just the right way. They then placed the whole package into E. coli bacteria, and checked which bacteria showed the ability to move when atrazine was present.
The bacteria that passed this test all turned out to carry the same switch sequence. Through further biochemical analysis of the RNA, Gallivan’s team showed that the switch works by preventing the cell’s protein production machinery from accessing the movement gene’s messenger RNA unless atrazine binding changes the shape of the switch and thereby frees the access point.
In the final step, the team also equipped the switch-carrying bacteria with an atrazine-degrading gene isolated from a different bacterium species. The resulting bacteria demonstrate their seek-and-destroy behavior by forming rings in petri dishes covered with atrazine as they move toward the atrazine and clear it from the plate.