Published: October 23rd, 2018
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.
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.
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 biomaterials8,9. 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.
1. Preparation of pDawn-Ag43 Bacterial Strains
2. Preparation of the Projector Optical Set-up for Illuminating Bacteria
3. Culturing Patterned Biofilms
4. Imaging Patterned Biofilms
5. Protocol Modifications/Alternatives
As seen in Figure 4A, pDawn-Ag43 bacteria were used to generate biofilms patterned in polystyrene well plates with projector illumination (the projector was set to illuminate a polka-dot pattern), imaged through brightfield microscopy with crystal violet stain as a contrast agent, and fluorescence microscopy using red-fluorescent-protein-expressing bacteria. Fluorescent biofilm samples can also be imaged using confocal microscopy14 to obtain images of the biofilm in 3-D (Figure 4B). In Figure 4C, we illustrate the high-resolution patterning possible by using a film photomask to provide patterned illumination to the biofilm sample. Finally, in Figure 4D and 4E, we demonstrate examples of patterning on glass and PDMS surfaces, as well as enclosed PDMS culture chambers—these illustrate the different types of environments where pDawn-Ag43 patterning can be applied.
Figure 1: Preparation of pDawn-Ag43 bacteria (protocol section 1). Preparing pDawn-Ag43 bacteria capable of light-regulated biofilm formation involves purifying pDawn-Ag43 plasmid from a host cloning strain, transforming it into an E. coli strain of interest, and creating bacterial freezer stock for long-term storage. Please click here to view a larger version of this figure.
Figure 2: Preparation of an optical set-up for biofilm sample illumination (protocol section 2). The optical set-up is housed inside a bacterial incubator and consists of a computer-connected projector illuminating a biofilm sample. Please click here to view a larger version of this figure.
Figure 3: Culture protocol for patterning biofilms (protocol section 3). (A) Prior to illumination, pDawn-Ag43 bacteria are prepared prior to patterning such that they are reliably induced at the proper growth phase. (B) After overnight illuminated growth, a patterned biofilm will be present at the bottom of the culture dish, along with planktonic cells in the liquid media, and after some further processing, the biofilm is ready for imaging. (C) As an alternative to well plates, biofilms can be cultured in enclosed culture chambers such as a molded PDMS cavity. In this case, syringes attached to blunt tip needles can be used to introduce the sample and flush liquids out of the chamber. (D) As an alternative to projector-based illumination patterns, patterns can also be generated by taping film photomasks directly to the bottom of biofilm culture chambers. In this case, the projector should be set up to illuminate blue light across the full field. Please click here to view a larger version of this figure.
Figure 4: Representative results of biofilms patterned using pDawn-Ag43. All results were obtained using an MG1655 E. coli host strain. (A) pDawn-Ag43 bacteria were used to generate biofilms patterned in polystyrene well plates with projector illumination (the projector was set to illuminate a polka-dot pattern), imaged through brightfield microscopy with crystal violet stain as a contrast agent, and fluorescence microscopy using red-fluorescent-protein-expressing bacteria. (B) Fluorescent biofilm samples are imaged with confocal microscopy to obtain 3-D images of the biofilm. (C) High-resolution biofilms can be patterned with a film photomask to provide patterned illumination to the biofilm sample. (D) Biofilms can be patterned on glass and PDMS surfaces. (E) Biofilms can be patterned in enclosed culture chambers. This figure has been adapted from previous work3. Please click here to view a larger version of this figure.
|Tranforming pDawn-Ag43 into host strain - no colonies
|Low plasmid concentration - check plasmid concentration on spectrometer. A typical miniprep of pDawn-Ag43 should yield at least 100 ng/μL; use up to 10-100 ng for transformation
|Check/remake competent cells: competent cells should have transformation efficiency at least 10^6 cfu/µg verified using a standard plasmid such as pUC19 - if not, remake competent cells
|Wrong (level of) antibiotic on LB agar plate - make sure to use 50 μg/mL spectinomycin for selection
|Projector illumination turns off / inconsistent overnight
|Disable problematic software such as: automatic overnight software/OS updating, night-time blue-light filter
|Projector may be overheating - set incubator to lower temperature while projector is turned on (e.g. 30 °C instead of 37 °C) - note projector as heat source can overheat incubator beyond set point
|Remove unnecessary sources of humidity from incubator, as these may affect projector electronics
|No/low levels of biofilm formed after overnight illumination, no planktonic cells growth either (i.e. liquid is clear)
|Wrong (level of) antibiotic - make sure to use 50 μg/mL spectinomycin
|Check everything is added to M63 recipe properly
|No/low levels of biofilm formed after overnight illumination, only planktonic cells (i.e. liquid is cloudy)
|Check light level, projector should be illuminating blue light at 50 μW/cm^2 at 460 nm wavelength
|Try letting bacteria grow for shorter/longer time after 1:1000 LB subdilution step prior to adding to M63
|Restreak bacteria on LB plate, start from fresh colony to generate overnight stationary phase culture
|Ensure projector is working consistently overnight - see point above
|Fuzzy biofilm patterns, high levels of background noise
|Reduce stray light from optical illumination system, cover reflective surfaces on interior of incubator
|Consider using photomask-based (as opposed to projector-based) structured illumination
|Check projector is properly focused at the bottom surface of the biofilm culture chamber
Table 1: Common troubleshooting issues.
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 patterning5,6, 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 biomaterials8,9 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 education20,21,22. 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).
The authors have nothing to disclose.
The authors thank D. Glass, H. Kim, N. Cira, A. Choksi, S. Rajan, and B. Keys for their helpful suggestions and the Spormann lab for access to their confocal microscope. Furthermore, the authors acknowledge the support from Stanford Bio-X Bowes and NSERC PGS fellowships, the National Institute of Health (R21-AI-139941), and the American Cancer Society (RSG-14-177-01).
|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
|MG1655 was used as E. coli strain of interest in this paper's representative results
|RFP expression plasmid
|Many options exist to obtain fluorescent bacteria - if using plasmid, ensure backbone does not conflict with colE1 ori of pDawn-Ag43
|Plasmid miniprep kit
|LB broth powder
|Add 20 g/L to water, autoclave, add 50 μg/mL spectinomycin to get sterile LB+spec
|LB agar powder
|Add 35 g/L to water, autoclave, add 50 μg/mL spectinomycin, pour into petri dishes to get sterile LB+spec plates
|Spectinomycin hydrochloride pentahydrate
|Make 1000x stock 50mg/mL in water, filter sterilize and dilute into media as needed
|Mix at 1:1 ratio with water, sterilize by autoclave or filter to obtain 50% glycerol
|M63 media salts 5X solution
|Add cas-amino acids, glucose and MgSO4, bring to 1X salts concentration by adding sterile water
|Make 20% stock in water, filter sterilize and add to M63 as supplement (final concentration 0.1%)
|Make 20% stock in water, filter sterilize and add to M63 as supplement (final concentration 0.2%)
|Make 1 M stock in water, autoclave and add to M63 as supplement (final concentration 1 mM)
|Dilute to 0.1% in water prior to use
|Self-hardening mounting media (Shandon immumount)
|Use to preserve samples over long term for fluorescence imaging
|Phosphate buffered saline (PBS) solution
|Can also use PBS prepared from powder / tablets
|6 well plate
|Used as biofilm culture dish for representative results
|Can be used to make enclosed microchamber cavities using soft lithography
|1 mL syringe
|For use with liquid handling with enclosed microchambers
|Blunt tip needle
|Attaches to 1 mL syringe
|Use to attach culture chamber to incubator ceiling
|Ensure interior height of incubator is tall enough to focus projector at the ceiling
|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
|Base for optical setup to hold projector - many other setups possible, just need to hold projector firmly at bottom of incubator, pointing upwards
|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
|Connects vertical and horizontal posts
|Connects optical breadboard base and vertical post
|Attaches vertical post to mounting base, mounting base to breadboard base
|Connects horizontal post to projector via tripod screw-hole
|Optical power meter
|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
|Connects to power meter (above) - UV detector not strictly necessary as blue light is within visible range
|Adjustable ND filter
|Adjustable (by rotating) neutral density filter - place above projector aperture
|Any software that allows drawing / presentation will suffice
|Used for designing photomasks, many mask printing services are compatible with AutoCAD files
|Many photomask printer services exist for high resolution (>30000DPI) film photomask printing
Copyright © 2024 MyJoVE Corporation. All rights reserved