High-resolution Patterned Biofilm Deposition Using pDawn-Ag43

We developed a method called biofilm lithography to shine light onto a surface, and bacteria accumulate where the light is. This method will allow us to answer key question in biofilm research, for example, how spatial structures affect community functions. There are many advantages to this method.

For example, it works at very high resolution of 25 micrometer, it does not require surface pre-patterning, and also works inside closed fluidic devices. Though this method can provide insight into the structure-function relationship of natural biofilms, it can also be applied to other disciplines, such as biofilm engineering, biomaterials, and multicellular synthetic biology. Visual demonstration of this method is critical as there are key preliminary steps to prepare the optical culture setup and bacterial sample prior to biofilm patterning.

First, transform pDawn-Ag43 plasmid into the E.coli strain of interest, and plate onto an LB agar plate supplemented with spectinomycin. Inoculate a single colony into LB broth with spectinomycin in a culture tube, and grow it to exponential phase in a shaking incubator at 250 rpm and 37 degrees Celsius. After inoculation, prepare a 25%glycerol stock of the pDawn-Ag43 transformed strain by mixing one milliliter of 50%sterile glycerol with one milliliter of culture in a cryo-tube.

Store the stock in a minus 80-degree Celsius freezer. Next, begin preparing the optical setup by placing a portable projector at the bottom of a bacterial incubator with the aperture pointing directly upward at the ceiling, where the biofilm culture chamber will be attached. Connect a laptop via the display cable to the projector inside the incubator.

Use tape to attach an empty dummy culture dish to the ceiling of the incubator. Adjust the focus on the projector by turning the focusing knob so that the focusing plane coincides with the bottom surface of the biofilm culture dish attached to the ceiling of the incubator. The projector should illuminate a sharp, non-blurry image onto the bottom of the culture dish.

Remove the dummy culture dish once the projection has been optimized. Using laptop software, direct the projector to illuminate the full field of view with maximum blue illumination by presenting a full blue slide. Measure the illumination intensity of the projector using an optical power meter by placing the photodetector head at the incubator ceiling and reading the intensity on the corresponding power meter calibrated to light wavelength of 460 nanometers.

Next, adjust the illumination intensity using an adjustable neutral density filter placed at the projector aperture. Rotate the filter to adjust the illumination intensity measured by the power meter until the illumination intensity in the center of the blue-light projected region reads 50 microwatts per centimeter squared. It is important to ensure the illumination intensity is set to a proper level using the calibrated photodetector positioned at the incubator ceiling.

This allows accurate tuning of the illumination intensity by rotating the neutral density filter. Now draw arbitrary patterns using the presentation projector software on the laptop, and display these patterns on the ceiling of the incubator, using the projector. Prior to illumination, streak a pDawn-Ag43 strain from the glycerol stock onto an LB-plus-spec agar plate.

Allow the colonies to grow overnight at 37 degrees Celsius. Inoculate a colony of pDawn-Ag43 bacteria from an agar plate in LB-plus-spec broth, and grow the culture overnight to stationary phase in a shaking incubator at 250 rpm and 37 degrees Celsius. On the following day, subdilute the culture at a ratio of one to 1, 000 in LB broth with spectinomycin.

Allow the subdiluted culture to grow in a shaking incubator at six hours at 250 rpm and 37 degrees Celsius until late exponential, early stationary phase. It is important to back-dilute the overnight culture and grow to late exponential phase prior to illumination. This ensures the bacteria are reliably healthy and active upon induction.

Following incubation, subdilute the late-exponential-phase culture at a ratio of one to 100 with previously prepared M63 media supplemented with 50-microgram-per-milliliter spectinomycin. Then, introduce the dilution into a biofilm culture dish. As the samples are now ready for illumination, tape the culture dish to the ceiling of the incubator, ensuring that the surface at the bottom of the dish is transparent for illumination from below by the projector.

After the overnight growth of the biofilm samples, remove the culture dish from the incubator. The dish will have biofilm bacteria attached to its bottom surface where it has been illuminated, as well as planktonic bacteria dispersed in the liquid media above. Discard the planktonic cells by removing the liquid media from the culture dish.

Next, rinse the sample two times with PBS to remove the remaining planktonic cells by gently pipetting in PBS, followed by aspiration. If the cells are fluorescently tagged, directly image the samples using fluorescence microscopy. As seen here, pDawn-Ag43 bacteria were used to generate biofilms patterned in polystyrene well plates with projector illumination.

Biofilms can be visualized using crystal violet staining or alternatively through fluorescence microscopy if using fluorescent bacteria. Fluorescent biofilm samples can also be imaged using confocal microscopy to obtain images of the biofilm in 3D. High-resolution patterning is possible by using a film photomask to provide patterned illumination to the biofilm sample.

Examples of patterning on glass and PDMS surfaces, as well as enclosed PDMS culture chambers, are shown here and illustrate the different types of environments where pDawn-Ag43 patterning can be applied. While attempting this procedure, it is important to first transform pDawn-Ag43 into your strain of interest, then properly focus and calibrate your projector, and finally follow the required culture and back-dilution steps to prepare your bacteria for illumination. Following this method, other techniques can be deployed, for example, monitoring biofilm long term under different culture conditions.

That way, we can understand how a biofilm structure affects its function. This technique will be widely applicable, for example, for synthetic biology and materials research, to engineer biofilms towards various applications.