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Bacterial biofilms represent a promising opportunity for engineering of microbial communities. However, our ability to control spatial structure in biofilms remains limited. Here we engineer Escherichia coli with a light-activated transcriptional promoter (pDawn) to optically regulate expression of an adhesin gene (Ag43). When illuminated with patterned blue light, longterm viable biofilms with spatial resolution down to 25 μm can be formed on a variety of substrates and inside enclosed culture chambers without the need for surface pretreatment. A biophysical model suggests that the patterning mechanism involves stimulation of transiently surface-adsorbed cells, lending evidence to a previously proposed role of adhesin expression during natural biofilm maturation. Overall, this tool--termed "Biofilm Lithography"--has distinct advantages over existing cell depositing/patterning methods and provides the ability to grow structured biofilms, with applications toward an improved understanding of natural biofilm communities, as well as the engineering of living biomaterials and bottom-up approaches to microbial consortia design. [ABSTRACT FROM AUTHOR]