Reprogramming Cells
 
Reprogramming Bacteria with Small Molecules and RNA
  Bacteria are terrific synthetic chemists—they can convert simple chemicals into highly complex natural products with efficiencies that are often significantly better than their human counterparts. Why do bacteria make complex natural products? The answer is simple: Bacteria depend on these molecules to grow faster than their competitors—the biosynthesis genes that direct the synthesis of these molecules give the cells an evolutionary advantage.
  We are developing evolutionary tools that allow us to “reprogram” bacteria to make new molecules. We began with a simple premise: “If a cell depends on a particular molecule to survive, the cell has a compelling reason to make it.” This is certainly true for essential metabolites such as amino acids, however, as chemists, we are interested in making new molecules that benefit us, but that may not benefit the cell directly. The challenge, then, is to make a cell depend on a new molecule for survival. This requires two things: a method of recognizing the molecule and a method of coupling this recognition event into a change in cell viability, perhaps by modulating gene expression.
  Riboswitches are RNA-encoded genetic control elements that regulate gene expression in a ligand-dependent fashion without the need for proteins. They are comprised of an aptamer domain, which recognizes the ligand, and an expression platform, which couples this binding event into a change in gene expression. Riboswitches have been shown to regulate a variety of metabolic pathways in prokaryotes and eukaryotes.
  In addition to natural riboswitches that control gene expression in response to endogenous metabolites, we and others have developed a variety of synthetic riboswitches that respond to non-endogenous small molecules. Because ligand recognition is performed by RNA aptamers that may be selected de novo using established techniques, it is possible to engineer riboswitches that respond to nearly any non-toxic, cell-permeable molecule that is capable of interacting with an RNA. As such, synthetic riboswitches have great potential to detect the production of small molecules in the context of directed evolution experiments, as well as to alter cell behavior in a variety of ways in a small molecule-dependent fashion.
  We recently created a synthetic riboswitch that couples the life of a bacterium to the presence of the small molecule theophylline. In these cells, theophylline activates the translation of an antibiotic resistance gene, while caffeine (a structurally-similar molecule) does not activate the resistance gene. When these cells are plated on the antibiotic chloramphenicol, they only grow when theophylline is present. Because these cells need theophylline for survival, but can’t make it themselves, they are auxotrophic. Auxotrophic cells hold an important place in bacterial genetics because they can be used to clone biosynthesis genes from other organisms. This ability to create “designer auxotrophs” that respond to new small molecules provides a powerful way to clone biosynthesis genes and direct the evolution of their protein products.
  More recently, we have shown that synthetic riboswitches can be used to control cell behavior. The movie below shows E. coli cells under a microscope. On the left, the cells do not move in the absence of a small molecule; addition of the small molecule causes the cells to move. We are currently reprogramming bacteria to follow new molecules for applications in bioremediation and medicine.