Bacteria routinely engage in “horizontal” gene transfer (HGT) to acquire new DNA sequences. Unlike “vertical” gene transfer, wherein genes are passed generationally from parent to offspring, DNA sequences can be transferred between species by direct cell-to-cell contact, by virus-mediated transfer, or by scavenging DNA from the environment. HGT by any of these mechanisms allows bacteria to rapidly acquire genes that provide new functions, such as antibiotic resistance or the ability to degrade anthropogenic pollutants during bioremediation. However, bacterial species have different genetic preferences for how they encode their genes, such that genes from one species may be unusable when transferred to another. Which properties make horizontally acquired genes incompatible, and why? I use a model system in which the bacterium E. coli depends on a horizontally acquired gene (glpK) to grow on glycerol as a sole carbon source. By testing glpKs from various species, I begin to identify genetic incompatibilities that prevent foreign gene expression. Understanding how genetic compatibility correlates with the function of horizontally acquired genes will help us understand barriers that prevent bacteria from gaining new functions by HGT.