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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.

Bacterial biofilms represent a promising opportunity for engineering of microbial communities. However our ability to control spatial structure in biofilms remains limited. Here we engineerEscherichia coliwith a light-activated transcriptional promoter to optically regulate adhesin gene expression. When illuminated with patterned blue light, long-term viable biofilms with spatial resolution down to 25μmcan be formed on a variety of substrates and inside enclosed culture chambers without the need for surface pretreatment. A biophysical model suggests the patterning mechanism involves stimulation of transiently surface-adsorbed cells, lending new 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 and patterning methods and provides the ability to grow structured biofilms, with applications towards an improved understanding natural biofilm communities, as well as the engineering of living biomaterials and bottom-up approaches to microbial consortia design.

Over the past decade, synthetic biology has developed increasingly robust gene networks within single bacterial cells, but relatively few systems have demonstrated engineered multicellular behaviour. In contrast, naturally existing terrestrial bacteria primarily live in complex surface-attached communities known as biofilms. Within these biofilms, multiple distinct microbial sub-populations form intricate spatial structures including colony co-localization and cell-cell co-aggregation. This spatial organization allows bacterial communities to achieve cooperative behaviours such as metabolic division-of-labour, and conversely, ecological interactions between different microbial subpopulations in turn influence the spatial patterning within the biofilm. Using optogenetic, metabolic, and cell-adhesion tools from synthetic biology, we are developing a biofilm culture platform that can generate optically patterned, metabolically interacting microbial communities. The platform will provide spatiotemporal control of both cell-surface and cell-cell attachment, tunable for both strength and specificity, as well as adjustable regulation of intercellular metabolic interactions. Taken together, these represent new tools to investigate how spatiotemporal patterning develops in biofilms and to engineer synthetic microbial consortia capable of complex tasks requiring biological division of labour.