High-resolution Patterned Biofilm Deposition Using pDawn-Ag43

1. Preparation of pDawn-Ag43 Bacterial Strains

1. Transform pDawn-Ag43 into an E. coli strain of interest (Figure 1).
   1. Grow a cloning strain hosting pDawn-Ag43 plasmid (obtainable from a plasmid repository) by inoculating the strain in LB broth supplemented with 50 μg/mL spectinomycin (LB+spec) in a culture tube (overnight in a shaking incubator at ~250 rpm, 37 °C). Then, use a miniprep kit to harvest the purified pDawn-Ag43 plasmid¹⁰.
   2. Choose an E. coli strain of interest to be patterned. Thus far, pDawn-Ag43 has been verified to work in MG1655³ and BW25113.
   3. Use an established protocol to generate competent (e.g., chemically competent¹¹ or electrocompetent¹²) stocks of the chosen E. coli strain (e.g., MG1655).
   4. Transform pDawn-Ag43 plasmid into the competent bacteria^11,12, followed by 1 h recovery, and plate on LB+spec agar plates. Allow colonies to grow overnight at 37 °C.
2. Store the pDawn-Ag43 transformed strains.
   1. Inoculate a single colony of pDawn-Ag43 from an LB+spec agar plate into LB+spec broth in a culture tube and grow it in a shaking incubator (at ~250 rpm, 37 °C) to exponential phase (OD600 ~0.4 – 0.8).
   2. Prepare stock to store the pDawn-Ag43 transformed strain at -80 °C long-term by mixing 1 mL of culture with 1 mL of 50% sterile glycerol in a cryo-tube to obtain a 25% glycerol freezer stock. Store this in a -80 °C freezer.
   3. If additional plasmids (e.g., fluorescent reporter plasmids) need to be transformed, create competent cells^11,12 from the pDawn-Ag43 transformed strain and repeat the transformation process^11,12 for additional plasmids as needed.
      NOTE: If using electroporation⁸, it is possible to cotransform multiple plasmids (including pDawn-Ag43) simultaneously; however, a simultaneous transformation is not recommended using chemical transformation methods, as the large size (> 10 kB) of pDawn-Ag43 means transformation efficiencies are reduced.

2. Preparation of the Projector Optical Set-up for Illuminating Bacteria

1. Obtain a bacterial incubator with non-transparent walls and a hole for passing cables, a portable projector capable of fitting and functioning inside the bacterial incubator, and a laptop equipped with software for presentation-projection (see Table of Materials). When choosing the projector and incubator, ensure that the minimal focus distance of the projector is less than the interior height of the incubator.
2. Place the projector inside the incubator at the bottom, with the aperture pointing directly upward, at the ceiling, where the biofilm culture chamber is attached (Figure 2).
3. Fix the projector in place by constructing a set-up with an optical breadboard base connected to a vertical post, in turn connected to a horizontal post which screws into the projector (Figure 2). Note that this set-up can be modified depending on the projector/available parts. Ultimately, the key requirement is that the projector is solidly fixed near the bottom of the incubator, with the aperture pointing upward.
4. Connect the laptop via the display cable (e.g., HDMI) to the projector inside the incubator.
5. If the surfaces—especially the ceiling—of the interior of the incubator are reflective (e.g., metal polished surface), cover them with dark matte surfaces to minimize reflections.
6. Use tape to attach an empty 'dummy' culture dish to the ceiling of the incubator. Note that, as with the projector, there are multiple acceptable ways to attach a culture dish; ensure that the transparent bottom surface of the culture chamber, where illumination occurs, is not covered.
7. 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 (e.g., a well plate) attached to the ceiling of the incubator (Figure 2). 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.
8. Using laptop software, direct the projector to illuminate the full field of view with maximum blue illumination (e.g., RGB = [0, 0, 255]) by presenting a full blue slide.
9. 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 460 nm. Follow the instructions on connecting and calibrating the photodetector for the specific power meter used.
   1. Reduce ambient light (e.g., turn off room lights, or place the incubator away from light sources) as much as possible prior to making illumination intensity measurements.
10. 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 μW/cm².
    NOTE: While it is possible to adjust the illumination intensity by using lower blue RGB values on the software end, using a filter while maximizing the blue RGB value has the advantage of maximizing the optical contrast of the system between illuminated vs. dark regions.
11. Draw arbitrary patterns using the presentation/projector software on the laptop and display these patterns on the ceiling of the incubator, using the projector.
    NOTE: Biofilm-forming regions should be drawn with maximum blue illumination (e.g., RGB = [0, 0, 255]), non-biofilm-forming regions with no illumination (e.g., RGB = [0, 0, 0]).

3. Culturing Patterned Biofilms

1. Prior to illumination, prepare the pDawn-Ag43 bacteria, starting from the glycerol freezer stock, so that they are reliably induced at the late exponential growth phase for illumination (Figure 3A).
   1. Streak a pDawn-Ag43 strain from glycerol stock onto LB+spec agar plates. Allow colonies to grow overnight (37 °C).
      NOTE: From here until the illumination culture step, ensure the cells stay in the dark as much as possible—brief periods at ambient illumination (e.g., for subdilution) are acceptable.
   2. Inoculate a colony of pDawn-Ag43 bacteria from an agar plate in LB+spec broth and grow the culture overnight to stationary phase (in a shaking incubator at ~250 rpm, 37 °C, for ~16 h).
   3. Subdilute the culture at a ratio of 1:1,000 with LB+spec broth (e.g., add 1 μL of overnight culture to 1 mL of fresh LB+spec).
   4. Allow the subdiluted culture to grow until late exponential/early stationary phase (OD ~1.0, ~6 h in a shaking incubator at ~250 rpm, 37 °C).
   5. While waiting for the culture to grow, prepare M63 media with 1x M63 salts, 1 mM MgSO₄, 0.2% glucose, 0.1% casamino acids, and 50 μg/mL spectinomycin in water (ensure the constituent parts are sterile).
   6. Subdilute the late-exponential-phase culture at a ratio of 1:100 with M63 media supplemented with 50 μg/mL spectinomycin. Then, introduce the dilution into a biofilm culture dish (e.g., pipette the diluted sample into a well plate).
      NOTE: The volumes required for this 1:100 subdilution depend on the culture dish being used (e.g., if using a 6-well polystyrene well plate [non-tissue-culture treated], a single sample would require adding 20 μL of culture to 2 mL of M63+spec, as the standard well volume of a 6-well plate is ~2 mL).
2. 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.
   NOTE: It is important to ensure that the ceiling of the incubator is not reflective, to minimize stray illumination. Stray illumination can also be reduced by using black-walled plates as culture dishes, although this is not strictly necessary—if using such plates, ensure the bottom surface is transparent.
3. Allow the biofilms to culture in the incubator overnight (16 h with no shaking, at 37 °C). Note that some projectors become less reliable at higher temperatures. If that is the case, culture at lower temperatures (e.g., 30 °C), keeping in mind that the incubation time may need to be increased, depending on the E. coli strain.

4. Imaging Patterned Biofilms

1. 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.
2. Discard the planktonic cells by removing the liquid media from the culture dish (e.g., by gently aspirating with a pipette).
3. Rinse the sample 2x with a phosphate-buffered saline (PBS) solution to remove the remaining planktonic cells (Figure 3B) by gently pipetting in PBS, followed by aspiration.
4. If the cells are fluorescently tagged, directly image the samples using fluorescence microscopy¹³ (e.g., wide-field¹³, 3-D confocal¹⁴, etc.).
   NOTE: Fluorescent biofilms can also be preserved using a self-hardening mounting medium. Apply one drop of mounting media to a biofilm sample, cover it with a glass coverslip, taking care not to capture any bubbles underneath, and allow it to harden overnight before imaging.
5. If the bacterial cells used are not fluorescently tagged, apply the crystal violet stain technique¹⁵ (Figure 3B) to enhance biofilm contrast prior to imaging.

5. Protocol Modifications/Alternatives

1. Grow pDawn-Ag43 bacteria on different surfaces.
   1. Put glass or poly-dimethyl-siloxane (PDMS) coupons (e.g., coverslips or thin strips of PDMS) into well plates prior to the addition of a bacterial sample/illumination, and follow the same protocol as before to pattern pDawn-ag43 biofilms on glass and PDMS.
2. Grow pDawn-Ag43 bacteria inside a transparent, enclosed culture chamber.
   1. Generate a culture chamber mold for PDMS.
      1. For a basic culture chamber, generate mold by attaching a hard, rectangular prism to a flat surface (the prism will become a cavity in the PDMS that serves as the culture chamber once the mold is cast). Note that more intricate culture chamber molds can be fabricated using soft lithography¹⁶.
   2. Cast a PDMS cavity by pouring PDMS into the mold and allowing it to cure (for a detailed soft lithography protocol, see JoVE's Science Education Database¹⁶).
   3. After curing, trim any excess PDMS, punch inlet/outlet channels into the cavity/culture chamber and bond the cavity to a flat surface (e.g., glass/polystyrene) by firmly pressing the PDMS onto the flat surface, leaving the cavity between the surface and the PDMS ceiling as the biofilm culture chamber.
      NOTE: More permanent bonding based on plasma treatment¹⁶ can also be used, but the chips will then not be reusable.
   4. Follow the culture protocol as before, using a syringe with blunt tip needles (instead of a pipette) to introduce the bacterial sample into the culture chamber/rinse with PBS buffer (Figure 3C).
      1. If using a temporarily bonded cavity, use only negative pressure to pull liquid into/out of the chamber, to ensure that the cavity does not become unbonded from the underlying glass/polystyrene surface.
3. Grow pDawn-Ag43 bacteria using a film photomask for structured illumination.
   1. Design a biofilm pattern using CAD software compatible with a film photomask printer/print service. The photomask film design should be clear in regions where the biofilm is meant to be printed, and black/opaque elsewhere. When complete, send the photomask file to the printer/printing service and await the return of the physical photomask.
   2. Instruct the projector to illuminate a full field of view with maximum blue illumination (e.g., RGB = [0, 0, 255]) using laptop software.
   3. Cut out a region of interest from the larger film photomask, and tape it directly to the bottom of the biofilm culture dish prior to introducing the bacterial sample for overnight illumination (Figure 3D). Culture the biofilms as before and remove the photomask after culturing, prior to imaging.