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Significance Bacteria live in surface-attached communities known as biofilms, where spatial structure is tightly linked to community function. We have developed a genetically encoded biofilm patterning tool (“Biofilm Lithography”) by engineering bacteria such that the expression of membrane adhesion proteins responsible for surface attachment is optically regulated. Accordingly, these bacteria only form biofilm on illuminated surface regions. With this tool, we are able to use blue light to pattern Escherichia coli biofilms with 25 μm spatial resolution. We present an accompanying biophysical model to understand the mechanism behind light-regulated biofilm formation and to provide insight on related natural biofilm processes. Overall, this biofilm patterning tool can be applied to study natural microbial communities as well as to engineer living biomaterials., 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, long-term 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.

Spatial structure and patterning play an important role in bacterial biofilms. Here we demonstrate an accessible method for culturing E. coli biofilms into arbitrary spatial patterns at high spatial resolution. The technique uses a genetically encoded optogenetic construct-pDawn-Ag43-that couples biofilm formation in E. coli to optical stimulation by blue light. We detail the process for transforming E. coli with pDawn-Ag43, preparing the required optical set-up, and the protocol for culturing patterned biofilms using pDawn-Ag43 bacteria. Using this protocol, biofilms with a spatial resolution below 25 μm can be patterned on various surfaces and environments, including enclosed chambers, without requiring microfabrication, clean-room facilities, or surface pretreatment. The technique is convenient and appropriate for use in applications that investigate the effect of biofilm structure, providing tunable control over biofilm patterning. More broadly, it also has potential applications in biomaterials, education, and bio-art.