Showing 1 - 3 of 3 results
1.
Optogenetic patterning generates multi-strain biofilms with spatially distributed antibiotic resistance.
Abstract:
Spatial organization of microbes in biofilms enables crucial community function such as division of labor. However, quantitative understanding of such emergent community properties remains limited due to a scarcity of tools for patterning heterogeneous biofilms. Here we develop a synthetic optogenetic toolkit 'Multipattern Biofilm Lithography' for rational engineering and orthogonal patterning of multi-strain biofilms, inspired by successive adhesion and phenotypic differentiation in natural biofilms. We apply this toolkit to profile the growth dynamics of heterogeneous biofilm communities, and observe the emergence of spatially modulated commensal relationships due to shared antibiotic protection against the beta-lactam ampicillin. Supported by biophysical modeling, these results yield in-vivo measurements of key parameters, e.g., molecular beta-lactamase production per cell and length scale of antibiotic zone of protection. Our toolbox and associated findings provide quantitative insights into the spatial organization and distributed antibiotic protection within biofilms, with direct implications for future biofilm research and engineering.
2.
High-resolution Patterned Biofilm Deposition Using pDawn-Ag43.
Abstract:
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.
3.
Biofilm Lithography enables high-resolution cell patterning via optogenetic adhesin expression.
Abstract:
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 (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.
