High-resolution Patterned Biofilm Deposition Using pDawn-Ag43

Summary

We demonstrate a method for depositing Escherichia coli bacterial biofilms in arbitrary spatial patterns with a high resolution using optical stimulation of a genetically encoded surface-adhesion construct.

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.

Introduction

Biofilms are surface-attached communities of microbes, and are well-known for their strong structure-function coupling. Spatial geometry and patterning of biofilms play an important role in overall community function (and vice versa)¹. The small length scales involved in biofilm structure—on the order of tens of microns²—make tunable and convenient control of biofilm patterning a challenging problem. Here we demonstrate a protocol that allows for biofilms to be precisely patterned in arbitrary geometries, based on optical illumination.

The protocol presented here uses pDawn-Ag43³, an optogenetic construct that couples biofilm formation in E. coli bacteria to optical illumination by driving the expression of Ag43 (an adhesin gene responsible for surface adhesion and biofilm formation) under the control of pDawn⁴ (a transcriptional regulator controlled by optical illumination). The method is convenient to use and can pattern biofilms on various surface environments, including enclosed (transparent) culture chambers. Compared to existing cell deposition methods, such as droplet-based deposition⁵ or surface prepatterning/treatment⁶, pDawn-Ag43 does not require microfabrication or clean-room facilities and does not require materials beyond those available to a typical microbiology laboratory. It is able to pattern with a spatial resolution below 25 μm, approaching the spatial dimensions of microcolonies in naturally existing biofilms². Overall, this technique provides the ability to manipulate biofilm structure, which then opens many avenues to study microecology in bacterial communities⁷. Additionally, patterned biofilms may provide a convenient platform upon which to engineer useful biomaterials^8,9. In this paper, we discuss the basic protocol required for patterning biofilms using pDawn-Ag43 and address potential modifications and troubleshooting related to the method.

Protocol

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.

Discussion

In light of the need for research tools that allow for biofilm structure control, we have presented an easy-to-use protocol for patterning bacterial biofilms using the pDawn-Ag43 optogenetic construct. With this technique, E. coli biofilms can be optically patterned on various surface environments, including enclosed chambers, with a spatial resolution below 25 μm.

Overall, this protocol can be broken down into four main sections: (1) the preparation of the pDawn-Ag43 bacteria, (2) the preparation of the optical and culture set-up hardware, (3) the pre-illumination bacterial growth steps, and (4) the post-illumination rinses and imaging.

The critical part of section 1 is the successful transformation of pDawn-Ag43 plasmid into the E. coli strain of interest. This is facilitated by isolating high-quality purified plasmid and generating high-quality competent cells for transformation (Table 1, troubleshooting).

The critical part of section 2 is the optimization of the projector set-up so that the illumination intensity is adjusted to 50 μW/cm² at the 460-nm wavelength, and the projector is properly focused at the biofilm sample height. Note that in this protocol, we describe an inverted illumination set-up where the projector shines light from below, upward toward the biofilm sample. The advantage of this set-up is that the light only needs to travel through the bottom of the culture dish before reaching the biofilm formation surface. Illumination from above means that the light would have to travel through the liquid media above the biofilm surface, which, during the course of the growth, gets cloudy with planktonic cells. In addition to these concerns, it is also important to minimize stray light in the optical set-up as much as possible, for example, by covering up reflective surfaces on the interior of the incubator—this helps to obtain sharper patterned biofilms. On a related note, sharper biofilm patterns can also be obtained by using a photomask to control illumination patterning (Figure 3D, Figure 4C). Common issues requiring troubleshooting include projector reliability issues at higher temperatures (e.g., 37 °C), which can be minimized by incubating the biofilm growth at lower temperatures (e.g., 30 °C), as well as computer software that causes operating system updates or blue light filtering during overnight growth (Table 1). It is also important to note that, depending on the projector and incubator model used, it is also possible that heat generated from the projector will result in a higher interior temperature than the incubator set temperature, which may need to be corrected.

The critical part of section 3 is obtaining reliable and repeatable bacterial samples before they are induced by illumination. For this reason, it is recommended to obtain clonal colonies of pDawn-Ag43 bacteria by streaking them out on an agar plate and then using the liquid culture steps to ensure that the bacteria are illuminated/induced at the late exponential growth phase in a repeatable manner.

Finally, the critical part of section 4 is to thoroughly, but also gently, wash away the planktonic cells remaining after the biofilm patterning protocol; thus, it is recommended to perform multiple gentle rinse steps with PBS.

Compared to existing techniques for cell patterning^5,6, optical biofilm patterning based on pDawn-Ag43 has a reasonably low barrier of entry to use, in that it does not require microfabrication, clean-room facilities, complex chemistry, or surface pretreatment, yet is still able to pattern with the high resolution (25 μm) typically associated with microfabrication techniques. The method extends previous work on bacterial photolithography for controlling gene expression¹⁷. Currently, pDawn-Ag43 plasmid is limited to E. coli, as it uses a pUC-based origin of replication, but pDawn and Ag43 are both compatible in other (Gram-negative) bacterial species. Genetic techniques are available for potentially introducing light-regulated biofilm formation to different bacterial species and represents a possible direction for future research. Another potential limitation of the technique is that it works by increasing biofilm formation in strains with weak native biofilm formation (e.g., MG1655 E. coli). However, strains with strong native biofilm formation have biofilms form regardless of illumination conditions, precluding patterned biofilm formation using pDawn-Ag43 as described here; yet optogenetic techniques may still prove applicable in regulating biofilm formation. We note that in other contexts, alternative methods of biofilm patterning may be available, such as via optical c-di-GMP modulation¹⁸.

Overall, pDawn-Ag43 based patterning will be appropriate for use in applications that investigate the effect of biofilm structure on function¹ and, therefore, could benefit from tunable control over biofilm patterning—a particularly relevant example to highlight is the study of microbial ecology in biofilms². Future directions include making patterned biomaterials^8,9 and/or structured bacterial communities. Alternative applications of this accessible protocol also include bio-art¹⁹, given the clear aesthetic potential, as well as formal and informal life science education^20,21,22. From an educational perspective, the protocol described here combines many relevant techniques (bacterial culture, transformation, optics/optogenetics) and is also modularly extendable (e.g., include microfluidics).

Materials

Name	Company	Catalog Number	Comments
DH5alpha/pDawn-Ag43	Addgene	107742	DH5alpha cloning strain hosting pDawn-Ag43 plasmid - plasmid needs to be moved to E. coli strain of interest prior to use
MG1655 E. coli	Coli Genetic Stock Center - Yale University	CGSC #6300	MG1655 was used as E. coli strain of interest in this paper's representative results
RFP expression plasmid	iGEM biobricks	J04450-pSB4K5bb	Many options exist to obtain fluorescent bacteria - if using plasmid, ensure backbone does not conflict with colE1 ori of pDawn-Ag43
Plasmid miniprep kit	Qiagen	27104
LB broth powder	Affymetrix	75852	Add 20 g/L to water, autoclave, add 50 μg/mL spectinomycin to get sterile LB+spec
LB agar powder	Affymetrix	75851	Add 35 g/L to water, autoclave, add 50 μg/mL spectinomycin, pour into petri dishes to get sterile LB+spec plates
Petri dishes	Fisherbrand	431760
Spectinomycin hydrochloride pentahydrate	abcam	ab141968	Make 1000x stock 50mg/mL in water, filter sterilize and dilute into media as needed
Glycerol	Sigma-Aldrich	G5516	Mix at 1:1 ratio with water, sterilize by autoclave or filter to obtain 50% glycerol
M63 media salts 5X solution	Bio-world	705729	Add cas-amino acids, glucose and MgSO4, bring to 1X salts concentration by adding sterile water
Casamino acids	Amresco	J851	Make 20% stock in water, filter sterilize and add to M63 as supplement (final concentration 0.1%)
D-glucose	Sigma-Aldrich	G8270	Make 20% stock in water, filter sterilize and add to M63 as supplement (final concentration 0.2%)
Magnesium sulfate	Sigma-Aldrich	M7506	Make 1 M stock in water, autoclave and add to M63 as supplement (final concentration 1 mM)
Crystal violet	Acros organics	212120250	Dilute to 0.1% in water prior to use
Self-hardening mounting media (Shandon immumount)	Thermo Scientific	9990402	Use to preserve samples over long term for fluorescence imaging
Phosphate buffered saline (PBS) solution	Gibco	10010023	Can also use PBS prepared from powder / tablets
6 well plate	Fisherbrand	351146	Used as biofilm culture dish for representative results
PDMS kit	Dow	SYLGARD 184	Can be used to make enclosed microchamber cavities using soft lithography
1 mL syringe	BD syringe	309659	For use with liquid handling with enclosed microchambers
Blunt tip needle	CML supply	901-23-050	Attaches to 1 mL syringe
Lab tape	Fisherbrand	15-901	Use to attach culture chamber to incubator ceiling
Bacterial incubator	Sheldon Manufacturing	SMI6	Ensure interior height of incubator is tall enough to focus projector at the ceiling
Portable projector	Ivation	IV-PJ-PRO-4-1	Many portable projector models exist, pDawn-Ag43 has been tested with multiple models including LED/laser based, with blue light channel ranging from 450-460nm central wavelength
Optical breadboard base	ThorLabs	MSB6	Base for optical setup to hold projector - many other setups possible, just need to hold projector firmly at bottom of incubator, pointing upwards
Optical post	ThorLabs	TR8	2 posts needed - one to be set up vertically extending out of breadboard base, one horizontally attached via right-angle clamp
Optical post right-angle clamp	ThorLabs	RA90	Connects vertical and horizontal posts
Mounting base	ThorLabs	BA1S	Connects optical breadboard base and vertical post
1/4"-20 screw	ThorLabs	SH25S050	Attaches vertical post to mounting base, mounting base to breadboard base
1/4"-20 set-screw	ThorLabs	SS25E63D	Connects horizontal post to projector via tripod screw-hole
Optical power meter	Newport	840	Use with power meter detector to measure projector illumination intensity - many power meter models exist, using one that has extendable detector will facilitate measurement
Optical power meter detector	Newport	818-UV	Connects to power meter (above) - UV detector not strictly necessary as blue light is within visible range
Adjustable ND filter	K&F Concept	SKU0689	Adjustable (by rotating) neutral density filter - place above projector aperture
Presentation-projector software	Microsoft	Powerpoint	Any software that allows drawing / presentation will suffice
CAD software	Autodesk	AutoCAD	Used for designing photomasks, many mask printing services are compatible with AutoCAD files
Film photomask	Fineline Imaging	n/a	Many photomask printer services exist for high resolution (>30000DPI) film photomask printing