Curli Fiber-Mediated Stabilization of Bacterial Hydrogel Patterns

Abstract

Source: Spiesz, E. M., et al., Three-dimensional Patterning of Engineered Biofilms with a Do-it-yourself Bioprinter. J. Vis. Exp. (2019)

This video demonstrates a method for comparing 3D-printed alginate patterns containing either control or genetically engineered bacterial strains. The test strain produces curli fibers upon induction, which reinforce the alginate hydrogel structure. Following treatment with a calcium-chelating solution, only the test pattern remains intact, highlighting the stabilizing effect of curli fiber production.

Protocol

1. 3D printing process

1. Install and open the 3D printing software (Table of Materials) on a computer. Connect the 3D printer to the computer. Move the printhead to its home position by clicking the home button for the X, Y, and Z axes.
2. For each print, place a prepared printing substrate onto a particular location on the printing bed.
3. Calibrate the height of the printhead in the Z axis.
   1. Raise the printhead to a height of 22 mm under manual control, so that it will not collide with the edge of the petri dish when moving to the desired position. Position the printhead overtop of the plate, and move it down until the pipette tip contacts the printing surface. Assign this Z-axis position as Z1 (the height of the printing surface).
   2. Raise the printhead, and move it outside of the plate area by manual control in the X, Y, and Z axes. If the working distance between the printhead and the plate surface is defined as Z2, enter Z1 + Z2 into the printing program as the Z-value during printing.
4. Program the printing shape by a self-developed point-by-point coordinate-determined method according to the desired trajectory.
   1. If the desired trajectory is a straight line, define only the start and end points. Including additional points on curved lines will result in smoother curves. Move the printhead manually to every point sequentially, and record the coordinates of these points in order. Enter all of these coordinates as well as the printhead moving speed for each printed segment into the G-code editor.
5. Both before and after printing, lift the printhead to a distance higher than the plate edge (20 mm), and move directly out of the plate region. Save this program as a G-code file and load directly for use in subsequent prints, while re-measuring the Z axis height for each new printing substrate.
   NOTE: See Table 1 for an example G-code for printing a square.
6. Load the pre-programmed G-code file. Open the G-code editor in the software, and program in the commands for printing the desired shape. At each command line, the position of the printhead may be changed in the X, Y, and/or Z axis. Input the Z value during all printing steps as Z1 + Z2 (height of printing surface + working distance).
   NOTE: The moving speed is also adjustable; 9,000 mm/min is a suitable value for typical printing rates.
7. Load the liquid bio-ink into syringe(s), and mount them in the syringe pump(s) of the 3D bioprinter.
8. Print the bio-ink onto the printing substrate by clicking the Print button.
9. During printing, control the printhead movement entirely by the software. Manually start the syringe pump before the printhead comes into contact with the printing surface.
   NOTE: The coordination of the syringe pump and the printer is empirically determined depending on the extrusion speed, the speed at which the printhead moves to the first print point, and the initial position of the printhead. If the initial printhead position is 20 mm, with a printhead speed of 9,000 mm/min and an extrusion speed of 0.1 mL/h, start the syringe pump immediately after the printing is started. If the extrusion speed is changed from 0.1 mL/h to 0.3 mL/h, then wait 2−3 s to start the syringe pump after the printing is started.
10. Stop the syringe pump as soon as the printhead arrives at the last point of printing. Halt the syringe pump before the printhead lifts up at the end of the printing process, otherwise excess bio-ink will drop onto the printing substrate and reduce the printing resolution.
11. For the construction of 3D structures, control all movements of the printhead in the G-code editor. Type in the printing height of the first layer. Increase the Z-value in the G-code by 0.2 millimeters for the second layer to increase the printing height. Thereafter, increase the Z-value by 0.1 millimeters when moving to a higher layer. Do not move the plate during the printing process.
12. To measure the width and height of the printed hydrogel, use a steel ruler placed underneath or alongside the sample.

2. Growing and testing the effectiveness of biofilm production by E. coli

1. Incubate the printed samples at room temperature for 3−6 days to allow the production of biofilm components (curli fibers). Image the plates using a camera or fluorescent scanner.
2. To dissolve the alginate matrix, add 20 mL of 0.5 M sodium citrate solution (pH = 7 adjusted with NaOH) to the printing substrates, and incubate for 2 h with 30 rpm shaking at room temperature. Discard the liquid and image the plates again to compare with the images of the plates before citrate treatment.

Table 1: Programming process and explanations of G-code for printing a square.

G-code commands	Tasks
G1 Z20 F9000	Lift the Z-axis to a height of 20 mm with a 9,000 mm/min moving speed.
G1 X95 Y65 F9000	Move to the starting point of the first line with a 9,000 mm/min moving speed.
G1 Z6 F9000	Move downwards in the Z-direction to a proper (here Z = 6 mm) printing distance.
G1 X95 Y105 F300	End point of the first line and starting point of the second line.
G1 X135 Y105	End point of the second line and starting point of the third line.
G1 X135 Y65	End point of the third line and starting point of the fourth line.
G1 X95 Y65	End point of the fourth line and starting point of the first line; a square is formed.
G1 Z20 F9000	Lift the Z-axis to a height of 20 mm at 9,000 mm/min.
G1 X55 Y40 F9000	Move to a coordinate (55, 40) outside of the Petri dish range.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LB broth powder	Sigma-Aldrich	L3022
Orbital shaker	VWR	89032-092	Model 3500
Petri dish	VWR	25384-326	150 x 15 mm
Rhamnose	Sigma-Aldrich	83650
Silicon tubing	VWR	DENE 3100103/25
Syringe pump	ProSense B.V.	NE-300
Sodium alginate	Sigma-Aldrich	W201502
Sodium citrate monobasic	Sigma-Aldrich	71498
Sodium hydrooxide	VWR	28244.295