Bioprinting living biofilms through optogenetic manipulation

Transcription

1 Supporting Information Bioprinting living biofilms through optogenetic manipulation Yajia Huang,, Aiguo Xia,, Guang Yang, *, and Fan Jin *, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, , PR China Hefei National Laboratory for Physical Sciences at the Microscale, Department of Polymer Science and Engineering, CAS Key Laboratory of Soft Matter Chemistry, University of Science and Technology of China, Hefei , PR China; * Corresponding author yang_sunny@yahoo.com or fjinustc@ustc.edu.cn. These authors contributed equally to this work. Table of Contents Additional Methods Page 2 Figure S1-S7 Page 3 9 Movie S1-S2 Page Table S1 Page 12 Reference Page 13 1 / 13

2 Additional Method Construction of Optogenetic and c-di-gmp Reporter Strain in P. aeruginosa Insertion mutant bphs with bpho or blrp1was constructed by mini-ctx or mini-tn7 system using a modified procedures for P. aeruginosa. 1, 2 We constructed unmarked insertion mutants by Flp-mediated excision of the antibiotic resistance marker. 3 First, bphs with bpho fragment obtained from the plasmid pind4-bphs 4 was cloned into the vector mini-ctx2 with the P A1/O4/O3 promoter in the upstream of MCS via a two-piece ligation, while blrp1 fragment obtained from gene synthesis in Sangon Biotech was cloned into the vector mini-tn7 with the constitutive promoter J23100 in the upstream of MCS via overlap PCR. The constructed plasmids were electroporated into PAO1 and constructed strain respectively and the corresponding recombinant strains via mini-ctx2 or mini-tn7 were identified by screening on LB agar plates containing 1mM IPTG and 100 μg ml -1 tetracycline or 30 μg ml -1 gentamycin. Then the strains were electroporated with a pflp2 plasmid and distinguished on LB agar plates containing 5% (w/v) sucrose for the excision of the resistance marker. The c-di-gmp reporter plasmid was constructed as the same procedures mentioned above. The c-di-gmp reporter plasmid was electroporated into bphs-blrp1 insertion mutant to monitor the intercellular c-di-gmp levels. The constructed plasmids and strains are listed on the Table S1. 2 / 13

3 Figure S1. Schematical showing the setup used in the bioprinting. Strain PAO1-bphS-blrP1-reporter was continuously cultured in a flow cell with a constant flow rate of 3.0 ml h -1. Meanwhile, four letters U S T C are microprojected onto the cover glass through a 20x oil objective, where 632 nm illuminations elevate the c-di-gmp levels that allows cells to attach to the surface, whereas 434 nm illuminations decrease the c-di-gmp levels that allows cells to detach from the surface. 3 / 13

4 Figure S2. Optimization of blue-illunimations for the bioprinting. Representative fluorescent images show how the strain PAO1-bphS-blrP1-reporter self-organized to form the T -pattern at the first 6 h using different power densities of blue illuminations ((a-d): 36.0 µw cm -2, 12.8 µw cm -2, 1.6 µw cm -2, 0 µw cm -2 ), where the red-illumination remain a constant power density (10.1µw cm -2 ). (e) Representative fluorescent images show how the strain PAO1-bphS-reporter self-organized to form the T -pattern at the first 6 h in the presence of a constant illumination of red-light (10.1 µw cm -2 ). Images with green or red color represent the images arising from sfgfp or CyOFP1 channel, respectively. Scale bar is 100 μm. 4 / 13

5 Figure S3. Optimization of red-illunimations for the bioprinting. Representative fluorescent images show how the strain PAO1-bphS-blrP1-reporter self-organized to form the T -pattern at the first 6 h using different power densities of red illuminations ((a-e): 26.6 µw cm -2, 10.1 µw cm -2, 5.0 µw cm -2, 2.7 µw cm -2, 0 µw cm -2 ), where the blue-illumination remained a constant power density (6.4 µw cm -2 ). Images with green or red color represent the images arising from sfgfp or CyOFP1 channel, respectively. Scale bar is 100 μm. 5 / 13

6 Figure S4. Optimization of projected pattern for the bioprinting. (a) Representative fluorescent images show how the strain PAO1-bphS-blrP1-reporter self-organized to form the T -pattern at the first 6 h using an inverted illumination, where blue-light ( 6.4 µw cm -2 ) was projected onto the letter and red-light was (10.1 µw cm -2 ) projected onto the complementary area. Representative fluorescent images show that the strain PAO1-reporter cannot self-organized to form the T -pattern at the first 6 h (b) in the presence of the illumination (red: 26.6 µw cm -2 and blue: 36 µw cm -2 ) or in the dark (c). Images with green or red color represent the images arising from sfgfp or CyOFP1 channel, respectively. Scale bar is 100 μm. 6 / 13

7 Figure S5. Representative bright-field images show the illuminated patterns of (a) red-light (632nm) or (b) blue-light (434nm) on a cover glass, where color maps indicated the averaged illumination densities used in the bioprinting. Scale bars in both panel a and b are 100 μm. 7 / 13

8 Figure S6. Optimization of inoculation cell densites for the bioprinting. Representative fluorescent images show how the strain PAO1-bphS-blrP1-reporter self-organized to form the T -pattern at the first 6 h using different inoculation cell densities ((a): cells cm 2, (b): cells cm 2, (c): cells cm 2 respectively), where the blue- and red-illumination remain a constant power density (blue: 6.4 µw cm -2 and red: 10.1 µw cm -2 ). Images with green or red color represent the images arising from sfgfp or CyOFP1 channel, respectively. Scale bar is 100 μm. 8 / 13

9 Figure S7. Optimization of circulating rate and carbron source for the bioprinting. Representative fluorescent images show how the strain PAO1-bphS-blrP1-reporter self-organized to form the T -pattern at the first 6 h using different circulating rate ((a): 3 ml h -1, (b): 5mL h -1 ) or different carbon source ((a,b): glutamate 0.6 mm, (c): succinate 0.6 mm), where the blue- and red-illumination remained a constant power density (blue: 6.4 µw cm -2 and red: 10.1 µw cm -2 ). Images with green or red color represent the images arising from sfgfp or CyOFP1 channel, respectively. Scale bar is 100 μm. 9 / 13

10 Movie S1. P. aeruginosa cells self-organized to form patterned biofilms in the presence of prescribed illuminations of U S T C during 6 h. Scale bar here is 100 μm. 10 / 13

11 Movie S2. 3D reconstructed image shows the successful bioprinting of living P. aeruginosa biofilms accorded to the prescribed illumination of T during 20 h. 11 / 13

12 Table S1. (Strains, plasmids, and primers used in this study) Description Source Strains PAO1 PAO1-reporter Wild type P. aeruginosa strain PAO1 with c-di-gmp reporter plasmid J.D. Shrout This study PAO1-bphS-blrP1 bphs and blrp1 derivative of PAO1 This study PAO1-bphS-blrP1-reporte r PAO1-bphS-reporter bphs and blrp1 derivative of PAO1 with c-di-gmp reporter plasmid bphs derivative of PAO1 with c-di-gmp reporter plasmid This study This study Plasmids mini-ctx2-bphs mini-tn7-blrp1 pucp20-pcdra-gfp-t-c yofp1 ptns2 pind4-bphs mini-ctx2 puc18t-mini-tn7t-gm pflp2 Tet r ; insertion of bphs with PA1/O4/O3 promoter constructed by PCR and cloned to HindIII/SpeI sites of mini-ctx2 Ap r, Gm r ; insertion of blrp1 with J23100 promoter constructed by PCR and cloned to HindIII/SpeI sites of mini-tn7 Gm r ; c-di-gmp reporter plasmid with an internal control based on pucp20 Ap r ; a helper plasmid for mini-tn7 site-specific transposition system plasmid carrying the bphs gene Tet r ; tet-frt-attp- MCS, ori, int, and orit Ap r, Gm r ; on mini-tn7t; orit on puc18 Ap r ; source of Flp recombinase This study This study 5 6, 7 4, 8, 9 1 2, 7 3 Primers Sequence bphs-hindiii-for 5 -GCCAAGCTTATGGCTAGAGGGTGCCTCAT-3 This study bphs-spei-rev 5 -GCCACTAGTTCCTTCATACCCGCCGGG-3 This study blrp1-hindiii-for 5 -GAGATAAGCTTTTGACGGCTAGCTCAGTCCT-3 This study blrp1-spei-rev 5 -GAGATACTAGTTTACAGGTCCATGGCCTGGC-3 This study sfgfp-hindiii-for 5 -GAGATAAGCTTTGGAAGGTTCCTTGGCGGCA-3 This study sfgfp-kpni-rev 5 -GAGATGGTACCTTATTTGTATAGTTCATCCATGCCATGTG- This study 3 CyOFP1-SacI-For 5 -GAGATGAGCTCGTATTAAAGAGGGGCGTGGG-3 This study CyOFP1-EcoRI-Rev 5 -GAGATCGAATTCAGGAAACAGCTATGACATGATTAC-3 This study 12 / 13

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