Optogenetics Enables High Resolution Biofilm Lithography

A new method allows researchers to shape the growth of bacterial communities by working with light and genetically engineered bacteria, creating intricate designs, from polka dots to stripes to circuits, overnight.

The technique can achieve biofilms grown at a resolution of 25 micrometers, which is about one-tenth the size of a grain of table salt.

“Most of the bacteria on Earth live in biofilm communities and biofilms are very relevant in disease in health—plaque on our teeth or catheter-based bacterial infection, for example. Understanding how biofilms function is an important question on many levels,”

said senior author Ingmar Riedel-Kruse, assistant professor of bioengineering at Stanford University.

Biofilm Lithography

The group’s technique relies on E. coli bacteria they have genetically engineered to secrete a sticky protein in response to a particular wavelength of blue light; the technique is based on optogenetics.

When they shine the appropriate wavelength light in the desired pattern on a culture dish of modified bacteria, the bacteria stick to the lit areas, forming a biofilm in the shape of the pattern. The researchers call their technique biofilm lithography for its similarity to lithography used in making electronic circuits.

Biofilm Lithography enables patterning with a lateral resolution down to 25 μm

(A) Confocal microscopy reveals average biofilm thickness of 15 μm, surface roughness coefficient of 0.33, and characteristic roughness length scale of 5.4 μm.
(B) Step-response analysis across light–dark boundary with high-resolution microscopy of striped illumination sample indicates ∼45μm spatial resolution; white triangles point to artifacts due to projector pixels.
(C) Using electrical tape as field stop (as opposed to a projector) enables a spatial resolution of ∼25μm.
(D) Biofilm patterned over large area and at high resolution are possible with photomask (shown example was originally designed for patterning microfluidic channels).
(E) Higher magnification imaging of D confirms feature sizes of ∼25μm; white triangle points to an example of a spurious individual cell.
(F) Schematic of Monte-Carlo simulation with cell swimming, adsorption/desorption, and light-regulated levels of adhesin A. In model 1, adhesin level is directly proportional to the adsorption rate of planktonic cells, whereas in model 2, adhesin level is inversely proportional to the desorption rate of adsorbed cells.
(G) No clear biofilm boundary can be observed at the light–dark border in model 1 where optically regulated adhesin production increases adsorption rate ka.
(H) Clear biofilm boundary can be observed in model 2 where optically regulated adhesin production decreases desorption rate kd. (For G and H, adsorbed cells are labeled red, illuminated region is marked in blue, and histogram of cell positions plotted below.)
Credit Xiaofan Jin, Ingmar Riedel-Kruse CC-BY

Other techniques for patterning bacterial communities exist, including depositing them with an inkjet printer or pre-patterning the culture surface with chemicals that bias bacterial growth in specific areas.

However, biofilm lithography has the benefit of speed, simplicity, higher resolution, and compatibility with a variety of surface environments including closed microfluidic devices, the researchers say.

Insights Into Bacterial Communities

The intricate designs made possible with biofilm lithography could help in exploring the dynamics of bacterial communities.

“Biofilms exist in a social environment with other bacteria. Interactions between these bacteria are often dictated by where they grow relative to each other and this could be a great tool for specifying exactly when and where in a bacterial community certain species can live,”

said Xiaofan Jin, a graduate student in bioengineering at Stanford and lead author of the paper.

While testing biofilm lithography, the researchers already happened upon a new insight. They had assumed that cells swimming in and out of illuminated regions would result in blurry patterns, but the designs turned out surprisingly sharp.

These crisp images led the group to conclude that many of the bacteria must already be weakly bound to the culture surface. Rather than cruising around the dish, it appears that bacteria are continuously jumping on and off the surface.

“In the literature, there are different models of how certain bacterial species form biofilms. We argue, at least with this species, that we provided additional evidence for that one hypothesis,”

explained Riedel-Kruse.

Conductive Biofilm Circuits

By coincidence, the 25 micrometer resolution the researchers achieved with biofilms is similar to the first silicon photolithography, which contributed to the widespread success of silicon semiconductors. Similarly, the researchers see many versatile and impactful applications for their bacterial designs.

“We’re hoping this tool can be applied toward further understanding bacterial communities, both natural and synthetic,” says Jin. “We also see potential in having these communities do useful things, such as metabolic biosynthesis or distributed biocomputation. It may even be possible to create novel biomaterials such as conductive biofilm circuits.”

The researchers are currently taking steps to grow multiple strains of bacteria simultaneously through biofilm lithography to make multi-species communities. In particular, they hope to understand how bacteria in a biofilm may share antibiotic resistance — a question with significant clinical implications, as biofilms are well-known for being stubborn against antibiotic treatment.

The work was supported by Stanford Bio-X, the Natural Sciences and Engineering Research Council of Canada, and the American Cancer Society.

Xiaofan Jin, Ingmar H. Riedel-Kruse
Biofilm Lithography enables high-resolution cell patterning via optogenetic adhesin expression
Proceedings of the National Academy of Sciences Mar 2018, 201720676; DOI: 10.1073/pnas.1720676115

Top Image: Xiaofan Jin/Ingmar Riedel-Kruse/Stanford