Orthogonal Ribosome Biofirewall

Transcription

1 Supporting Information Orthogonal Ribosome Biofirewall Bin Jia,, Hao Qi,, Bing-Zhi Li,, Shuo pan,, Duo Liu,, Hong Liu,, Yizhi Cai, and Ying-Jin Yuan*, Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin , PR China SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin , PR China 13 School of Biological Sciences, University of Edinburgh, Daniel Rutherford Building G.24, The King s Buildings, Edinburgh EH9 3BF, United Kingdom

2 Table S1. Strains and plasmids used and developed in this work. Figure S1. Complementary base pair interactions between the SD sequence of mrna and the ASD sequence of rrna. Figure S2. Secondary structure of the orthogonal 16S rrna. Figure S3. Orthogonal PCR tag of O-16S rrna for quantitative PCR. Figure S4. Analysis of the leakage of the orthogonal system. Figure S5. Growth of cells harboring O-ribosome systems. Figure S6. Detection of antibiotic-resistant microbes in nonsterile soil medium. Figure S7. Relative copy number assay of the O-rRNA plasmids in lab medium and nonsterile soil medium. Figure S8. Plasmid map of pjb2soribo2. Figure S9. Plasmid map of pjborfp. Figure S10. Plasmid map of pjb413c. Figure S11. Plasmid map of pjbvioabe. Figure S12. Plasmid map of pjbnot1. Figure S13. Plasmid map of pjbnot2. Figure S14. Plasmid map of pjb1scei with one I-SceI site. Figure S15. Plasmid map of pjb2scei with two I-SceI sites. Supplementary References

3 43 44 Table S1, Strains and plasmids used and developed in this work. Plasmid/Strain Description Reference pjbea Ampr, pmb1ori, psb1a3 control vector This study pjbec Cmr, p15a, psb3c5 control vector This study pjbribo Ampr, pmb1ori, pbad-rrna This study pjboribo1 Ampr, pmb1ori, pbad-o-rrna This study pjboribo2 Ampr, pmb1ori, pbad-o-rrna with modified loop This study pjborfp Cmr, p15a, plac- O-RFP This study pjb413c Cmr, p15a, His3, CEN/ARS, yeast assembly vector This study pjbvioabe pjb413c derivative containing plac-vioa-viob-vioe This study pjbovioabe pjb413c derivative containing plac-ovioa-oviob-ovioe This study pjbnot1 Kanr, psc101 replicon ts, ptac-ci-terminator-pr-rfp This study pjbnot2 Kanr, psc101 replicon ts, ptac-ci-terminator-pr-i-scei This study pjb1scei Ampr, pmb1ori, 1 SceI site, ptrc-rfp This study pjb2scei Ampr, pmb1ori, 2 SceI sites, ptrc-rfp This study pjb2soribo2 pjb2s derivative containing pbad-o-rrna with modified This study loop pkd46 Ampr, psc101 replicon ts, pbad-gam-beta-exo 44 MG1655Z1 F-, λ-, SpR, lacr, tetr 41 EJB000 MG1655Z1 containing pjbec and pjbea This study

4 EJB001 MG1655Z1 containing pjborfp and pjbea This study EJB002 MG1655Z1 containing pjboribo1 and pjborfp This study EJB010 MG1655Z1 containing pjboribo2 and pjborfp This study EJB020 MG1655Z1 containing pjbvioabe This study EJB030 MG1655Z1 containing pjbovioabe This study EJB040 MG1655Z1 containing pjboribo2 and pjbovioabe This study EJB041 MG1655Z1 containing pjbea EJB050 MG1655Z1 containing O-lacZ mutation in the genome This study EJB060 EJB05 containing pjboribo2 This study EJB070 MG1655Z1 containing pjbnot1 integrated into the genome This study EJB080 MG1655Z1 containing pjbnot2 integrated into the genome This study EJB090 EJB08 containing pjb1scei This study EJB100 EJB08 containing pjb2scei This study EJB110 EJB08 containing pjb2soribo2 and pjbovioabe This study 45 46

5 47 ASD UCCUCC Ribosomes AGGAGG AUG SD Figure S1. Complementary base pair interactions between the SD sequence of mrna and the ASD sequence of rrna. The two key elements for translation were the Shine-Dalgarno (SD) sequence and the anti-shine-dalgarno (ASD) sequence. The SD sequence is also known as the ribosome binding site (RBS), located approximately 6 12-bp upstream of the start codon AUG in the mrna. The ASD sequence lies at the 3' end of the 16S ribosomal RNA (rrna). Complementary base pair interactions occur between the SD sequence of mrna and the ASD sequence of rrna, guiding the 30S ribosomal subunit to the correct position for initiation process of translation. 58

6 O-ASD 3 loop O-16S rrna Figure S2. Secondary structure of the orthogonal 16S rrna. The green box indicates the orthogonal ASD site at the 3 end. The red box indicates the apex loop at nucleotides 81-89, which was switched to a modified loop to act as the orthogonal PCR tag. 64

7 a Target of orthogonal 16S Target of 23S rrna (Reference) Sample of Password system A P1 O-16S rrna P2 B P3 23S rrna P4 Sample of WT system C P1 WT-16S rrna D P3 23S rrna P2 P4 b 2log A B C D A B C D A B C D A B C D 2log Figure S3. Orthogonal PCR tag of O-16S rrna for quantitative PCR. (a) Primer P1 was designed to base pair with the modified loop while not binding the WT 68 loop P2 was downstream of this loop and base paired with the 16S rrna. P3 69 and P4 base paired with the 23S rrna. (b) Agarose gel electrophoresis of orthogonal 70 PCR tag. Different annealing temperatures were optimized for quantitative PCR

8 Figure S4. Analysis of the leakage of the orthogonal system. EJB000 is E. coli MG1655Z1 containing control vector pjbea and pjbec; EJB001 is E. coli MG1655Z1 containing pjbea and pjborfp; EJB010 is E. coli MG1655Z1 containing pjboribo2 and pjborfp. Cells were grown in LB media containing 1 mm IPTG and ampicillin and chloramphenicol for 24 h, and then culture samples were diluted to 0.5 of OD600 and the fluorescence detected on the Spectramax M2 (Molecular Devices) with excitation at 584 nm and measurement of the emission at 617 nm. The experiment was performed in triplicate. Error bars are SD. (Student t-test: n.s., not significant; *P = 0.05, **P = )

9 OD Control WT-Ribo O-Ribo Time (h) Figure S5. Growth of cells harboring O-ribosome systems. Cells were subcultured to OD600 of 0.1 in fresh LB media with Ara (10 mm) and grown for 10 h. Control and WT refer to cells harboring the empty expression vector and the wild-type rrnb operon, respectively. 90

10 LB agar LB-Amp agar 91 LB-Kan agar LB-CmR agar Figure S6. Detection of microbes in nonsterile soil medium. Before culturing E. coli containing the biofirewall, 200-µL samples of soil-water medium were plated on LB agar without antibiotics or containing ampicillin, kanamycin or chloramphenicol. The presence of microbe colonies with different colors and sizes on LB agar and LB-Amp agar indicated the presence of additional ampicillin-resistant microbes in the soil used, while those microbe colonies were sensitive to kanamycin and chloramphenicol

11 500bp a b LB-IA medium Nonsterile soli medium M LB-IA medium Nonsterile soli medium Figure S7. Relative copy number assay of the O-rRNA plasmids in lab medium and nonsterile soil medium. Single colonies were inoculated in 20 ml of LB-IA medium (LB containing 1 mm and 10 mm Ara) and nonsterile soil medium. (a) PCR analysis of the O-rRNA plasmid in medium. 1 ml culture of 12h, 24h, 36h, 48h, 60h were collected by centrifugation (12000 rpm, 5 min) and then filtrated with a 0.22 μm aqueous membrane, respectively. PCR analysis of those sterile cultures by P5 and P6, and analysis of PCR products by electrophoresis on a 1% agarose gel. (b) Relative copy number assay of the O-rRNA plasmids. 1 ml culture samples were collected at 24 h and 48 h, respectively. Total DNAs were extracted from cells by boiling the samples for 15 min, freezing at -80 C for 15 min, and then boiling again for another 15 min. After spinning at 15,000 rpm for 5 min, the resulting supernatant containing the total DNA was diluted appropriately and used for qpcr analysis. Genomic DNA containing only one copy of the O-rRNA was diluted and used as a standard curve. Quantitative PCR reactions (20 μl total) containing 1 μl of either sample or standard DNA template were prepared in accordance with the manufacturer s specifications. Primers P1 and P2 were used to detect O-rRNA plasmids. Primers P7 and P8 were

12 used to target the chromosomal lacz as reference gene. The copy number of O-rRNA plasmids were used to present the data of the O-rRNA plasmids relative to the internal control gene the lacz (the 2- Ct method 45 ) Figure S8. Plasmid map of pjb2soribo2.

13 Figure S9. Plasmid map of pjborfp Figure S10. Plasmid map of pjb413c. 130

14 Figure S11. Plasmid map of pjbvioabe Figure S12. Plasmid map of pjbnot1.

15 Figure S13. Plasmid map of pjbnot Figure S14. Plasmid map of pjb1scei.

16 Figure S15. Plasmid map of pjb2scei Supplementary References (41) Cox, R. S., 3rd, Dunlop, M. J., and Elowitz, M. B. (2010) A synthetic three-color scaffold for monitoring genetic regulation and noise. J Biol Eng 4, (43) Datsenko, K. A., and Wanner, B. L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U. S. A. 97, (45) Schmittgen, T. D., and Livak, K. J. (2008) Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3,