The genetic code of all living organisms is based on the sequence of the four nucleobases adenine, thymine, cytosine and guanine, which form paired bonds (G·C and A·T) in the DNA double helix. Now, Malyshev et al. report the generation of the first semi-synthetic microorganism that stably replicates an unnatural base pair (UBP). Expression of an algal nucleotide triphosphate transporter (NTT) in Escherichia coli cells enabled the direct uptake of the triphosphates of the nucleobases d5SICS and dNaM, which were previously shown to form an UBP. NTT-expressing cells that carried a plasmid containing the d5SICS·dNaM UBP were grown in medium that was supplemented with the two unnatural triphosphates. Importantly, the authors showed that the UBP was retained in the plasmid following days in culture and was replicated with high fidelity, possibly by polymerase I. The construction of a bacterium that encodes an extra base pair provides a platform to engineer cells with a vastly expanded genetic alphabet to increase biological diversity.