A genetically encoded system for programmable cell assembly using surface-displayed DNA oligonucleotides

Precise patterning of cells in three dimensions is highly desirable in both basic and applied research for tissue engineering. Although bottom-up cell assembly of with defined spatial arrangements has been achieved using cell-surface-bound DNA oligonucleotides, the current approaches require complicated chemical functionalization protocols, which is expensive, time-consuming and difficult to scale up. To overcome these limitations, we devised a genetically encoded system to attach customized single-stranded DNA (ssDNA) sequences onto Escherichia coli cell surface. In particular, in vivo produced ssDNA molecules containing a specific recognition site could be covalently bound to a phage protein, X174 A*, which was fused with a membrane protein as the cell surface anchor. As proof-of-concept, recombinant expression cassettes of X174 A* and the ssDNA biosynthesis machinery were constructed and introduced in E. coli, and the presence of surface-attached ssDNA molecules was examined using flow cytometry. Employing a cell-impermeable ssDNA-binding fluorescence dye, we observed fluorescence signals only when both X174 A* and ssDNA-synthesizing proteins were expressed. In addition, fluorescence was diminished after treatment using a ssDNA-specific exonuclease, confirming the successful display of ssDNAs on the cell surface. The feasibility of using this system for programmable assembly of E. coli cells is under investigation.IGBOpe