Name: three-dimensional patterning of engineered biofilms with a do-it-yourself bioprinter
Uploaded: 2019-05-16T14:00:00
Description: Watch this Scientific Journal Video about Three-dimensional Patterning of Engineered Biofilms with a Do-it-yourself Bioprinter at JoVE.com

This work was supported by an AOARD grant (No. FA2386-18-1-4059), the Netherlands Organization for Scientific Research (NWO/OCW) as part of the Frontiers of Nanoscience program, and the Advanced Materials NWO-NSFC program (No. 729.001.016). The authors acknowledge laboratory assistance of Ramon van der Valk and Roland Kieffer.

1. Conversion of a commercial 3D printer into a 3D bioprinter
<ol>
	<li>Remove the extruder and the heater of a commercial 3D printer (Table of Materials) from the printer frame, and unplug the wiring controlling these elements from the main circuit board (Figure 1A). Since the sensor that controls the operational temperature of the printer needs to be functional to communicate with the printer software, remove from the printing software the algorithm that delays printing ...

<ol>
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G., Yang, G., Jin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprinting+Living+Biofilms+through+Optogenetic+Manipulation.">Bioprinting Living Biofilms through Optogenetic Manipulation.</a> ACS Synthetic Biology. 7 (5), 1195-1200 (2018).</li><li>Jin, X. F., Riedel-Kruse, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+Lithography+enables+high-resolution+cell+patterning+via+optogenetic+adhesin+expression.">Biofilm Lithography enables high-resolution cell patterning via optogenetic adhesin expression.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (14), 3698-3703 (2018).</li><li>Stewart, P. S., Franklin, M. 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The protocol presented here for 3D printing of engineered biofilms has two critical steps. First is the preparation of the agar printing surface, which is the most critical factor to producing a specific printing resolution. It is important to ensure that the printing surface is flat and that the pipette tip on the printhead is positioned at the correct height from the surface. If the surface is not flat, the working distance will change during the printing process. If the working distance is less than 0.1 mm, the CaCl<s...

There is currently an increasing need to develop environmentally-friendly, sustainable solutions for the production of spatially-patterned materials, due to the expanding number of markets for such materials1. This article presents a simple, economical method for the production of such materials and therefore offers a large spectrum of future applications. The method presented here allows three-dimensional (3D) printing of spatially-patterned structures using a bio-ink containing living bacteria. Bacteria remain viable within the printed structures for over one week, enabling the bacteria to perform natural or engineered metabolic activities. Printed bacteria can thereby produce and deposit desired components within the printed structure, for example creating a functional cross-linked biofilm2.
Traditional methods for the production of advanced materials involve high energy expenditures (e.g., high temperatures and/or pressures) and can produce large quantities of chemical waste, often toxic substances that require cost-extensive utilization3,4. In contrast, multiple bacterial species are able to produce materials that can be readily applicable in various industries. These materials include polymers such as polyhydroxyalkanoates (PHA)5 or poly(glycolide-co-lactide) (PGLA)6, bacterial cellulose7, bacterial concrete materials8, biomimetic composites9, amyloid-based adhesives10, or bio-based electrical switches11, among others. Moreover, bacterial production of valuable materials typically takes place at near-ambient temperatures and pressures and in aqueous environments, without requiring or producing toxic compounds. While producing materials with bacteria has been demonstrated in the literature and some industrial applications have already emerged12,13, a reliable method for spatial patterning of such materials remains a challenge.
This article demonstrates a straight-forward method of converting a low-cost commercial 3D printer into a 3D bacterial printer. The protocol shows how to prepare a bio-ink containing and sustaining the living bacteria, as well as how to prepare substrates onto which the 3D printing can be performed. This method is appropriate to use with a variety of natural and engineered bacterial strains able to produce materials. These bacteria can be spatially distributed within a 3D printed structure and still continue their metabolic activity, which will result in a spatial distribution of the desired materials produced by the bacteria.
This printing method enables additive manufacturing of biofilms, aggregates of bacteria surrounded by a self-produced extracellular matrix. Biofilms are heterogeneous 3D networks in which proteins, polymers, bacterial cells, oxygen, and nutrients are all spatially structured14. While in the form of a biofilm, bacteria exhibit an increased antibiotic resistance and structural robustness, making them difficult to eradicate from surfaces including medical catheters and implants. The key to biofilm properties, and also the largest challenge to biofilm research, seems to be the heterogeneity of biofilms15,16,17. Production of spatially-controlled model biofilms is of special interest as it would allow for either reproducing or tuning the spatial patterns of biofilm components, aiding the understanding of the stable deposition of biofilms on virtually any surface in nature.
This article presents a method for the production of biofilms using 3D-printed hydrogels containing engineered E. coli bacteria that produce biofilm proteins in the presence of an inducer, as well as methods of verification of biofilm formation2. The major extracellular matrix components of these biofilms are curli amyloid fibers18 that contain self-assembled CsgA proteins. When engineered E. coli bacteria are induced to express CsgA proteins, they form a stable model biofilm that protects the cells against being washed off of the printing surface. Such a 3D printed biofilm can be spatially controlled and can serve as a useful research tool for the investigation of multiscale biofilm structure-function mechanics or materiomics19. These bespoke biofilms will aid the understanding of the principles of biofilm formation and their mechanical properties, enabling further research into the mechanisms of antibiotic resistance among other applications.

<table>
	<tbody>
		<tr>
			<td>3D printer</td>
			<td>CoLiDo</td>
			<td>3D-P Kit</td>
			<td></td>
		</tr>
		<tr>
			<td>3D printing software</td>
			<td>CoLiDo</td>
			<td>Print-Rite ColiDo Repetier-Host v2.0.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>05040</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>3886.1</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth powder</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>VWR</td>
			<td>89032-092</td>
			<td>Model 3500</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>VWR</td>
			<td>25384-326</td>
			<td>150 x 15 mm</td>
		</tr>
		<tr>
			<td>Rhamnose</td>
			<td>Sigma-Aldrich</td>
			<td>83650</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon tubing</td>
			<td>VWR&#160;</td>
			<td>DENE 3100103/25</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>ProSense B.V.&#160;</td>
			<td>NE-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium alginate</td>
			<td>Sigma-Aldrich</td>
			<td>W201502</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>71498</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydrooxide</td>
			<td>VWR</td>
			<td>28244.295</td>
			<td></td>
		</tr>
	</tbody>
</table>

three-dimensional patterning of engineered biofilms with a do-it-yourself bioprinter

Biofilms are aggregates of bacteria embedded in a self-produced spatially-patterned extracellular matrix. Bacteria within a biofilm develop enhanced antibiotic resistance, which poses potential health dangers, but can also be beneficial for environmental applications such as purification of drinking water. The further development of anti-bacterial therapeutics and biofilm-inspired applications will require the development of reproducible, engineerable methods for biofilm creation. Recently, a novel method of biofilm preparation using a modified three-dimensional (3D) printer with a bacterial ink has been developed. This article describes the steps necessary to build this efficient, low-cost 3D bioprinter that offers multiple applications in bacterially-induced materials processing. The protocol begins with an adapted commercial 3D printer in which the extruder has been replaced with a bio-ink dispenser connected to a syringe pump system enabling a controllable, continuous flow of bio-ink. To develop a bio-ink suitable for biofilm printing, engineered Escherichia coli bacteria were suspended in a solution of alginate, so that they solidify in contact with a surface containing calcium. The inclusion of an inducer chemical within the printing substrate drives expression of biofilm proteins within the printed bio-ink. This method enables 3D printing of various spatial patterns composed of discrete layers of printed biofilms. Such spatially-controlled biofilms can serve as model systems and can find applications in multiple fields that have a wide-ranging impact on society, including antibiotic resistance prevention or drinking water purification, among others.

The first step for successful 3D printing of biofilms is converting a commercial 3D printer into a bioprinter. This conversion is achieved by removing the extruder and heater of the printer, designed for printing with a polymeric ink, and replacing these with components appropriate for printing bio-ink containing living bacteria (Figure 1A). The extruder is replaced by a pipette tip (or tips, if multiple bio-inks will be used in the printing process) attached...

Watch this Scientific Journal Video about Three-dimensional Patterning of Engineered Biofilms with a Do-it-yourself Bioprinter at JoVE.com

Three-dimensional Patterning of Engineered Biofilms with a Do-it-yourself Bioprinter

This article describes a method of transforming a low-cost commercial 3D printer into a bacterial 3D printer that can facilitate printing of patterned biofilms. All necessary aspects of preparing the bioprinter and bio-ink are described, as well as verification methods to assess the formation of biofilms.

Biofilms are aggregates of bacteria embedded in a self-produced spatially-patterned extracellular matrix. Bacteria within a biofilm develop enhanced ...

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