Supporting Data for: A modular strategy for engineering orthogonal chimeric RNA transcription regulators 1
Melissa K. Takahashi and Julius B. Lucks 1
1*
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USA
* To whom correspondence should be addressed. Tel: 1-‐607-‐255-‐3601; Fax: 1-‐607-‐255-‐9166; Email:
[email protected] Materials and Methods Flow cytometry Ten µL of cell culture from the in vivo gene expression assay was mixed with 500 µL PBS and read on a BD FACSCalibur flow cytometer. Data for the following parameters were collected: forward scatter (FSC), side scatter (SSC), and GFP fluorescence (488 nm excitation, 530 nm emission). Data for at least 50,000 cellular counts were collected for each sample. Data for each plasmid combination were analyzed in matlab. Counts were first gated in FSC vs. SSC by choosing a window surrounding the largest cluster of cells. GFP values recorded in relative channel number (1-4096 corresponding to 12-bit data) for gated cells were then histogrammed in 500 bins. Histograms were converted into relative intensity values (010,000 for log4 mode acquisition) and plotted in Figure S9. Induction curve assay All experiments were performed in E. coli strain TG1. Plasmid combinations were transformed into chemically competent E. coli TG1 cells, plated on Difco LB+Agar plates containing 100 µg/mL carbenicillin and 34 µg/mL chloramphenicol, and incubated overnight (approximately 17 hours (h)) at 37°C. Plates were taken out of the incubator and left at room temperature for approximately 7 h. Three colonies were used to separately inoculate 300 µL of LB containing carbenicillin and chloramphenicol at the concentrations above in a 2 mL 96-well block (Costar 3960), and grown approximately 17 h overnight at 37°C at 1,000 rpm in a Labnet Vortemp 56 bench top shaker. 10 µL of this overnight culture was then added to 190 µL (1:20 dilution) of M9 minimal media (1xM9 minimal salts, 1 mM thiamine hydrochloride, 0.4% glycerol, 0.2% casamino acids, 2 mM MgSO4, 0.1 mM CaCl2) containing the selective antibiotics and the required amount of IPTG. After 3 h incubation at 37C, 1,000 rpm in the Vortemp shaker, 20 µL of this culture was diluted again into 180 µL (1:10 dilution) of supplemented M9 minimal media with antibiotics and IPTG, and grown for 2.5 h at 37C, 1,000 rpm. 100 µL of this culture was then transferred to a 96-well plate (Costar 3631) containing 100 µL of phosphate buffered saline (PBS). Fluorescence (485 nm excitation, 528 nm emission) and optical density (OD, 600 nm) were then measured using a Biotek SynergyHT plate reader. mRFP in vivo gene expression assay
1
Transformation and growth was performed according the methods for the in vivo gene expression assay outlined in the main text. Fluorescence (560 nm excitation, 630 nm emission) and optical density (OD, 600 nm) were measured using the Molecular Devices plate readers Spectra Max Gemini and Spectra Max 190 respectively.
Table S1: Important DNA sequences Name Sequence Super folder ATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATT green AGATGGTGATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGT fluorescent GATGCTACAAACGGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTA protein CCTGTTCCGTGGCCAACACTTGTCACTACTCTGACCTATGGTGTTCAATGCTT (SFGFP) TTCCCGTTATCCGGATCACATGAAACGGCATGACTTTTTCAAGAGTGCCATGC CCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGACCTAC AAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGA GTTAAAGGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAACTCG AGTACAACTTTAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAAT GGAATCAAAGCTAACTTCAAAATTCGCCACAACGTTGAAGATGGTTCCGTTCA ACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTT TACCAGACAACCATTACCTGTCGACACAATCTGTCCTTTCGAAAGATCCCAAC GAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTAC ACATGGCATGGATGAGCTCTACAAA Ribosome AGGAGGAAGGATCT binding site (RBS) TrrnB GAAGCTTGGGCCCGAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCC GTCGACCATCATCATCATCATCATTGAGTTTAAACGGTCTCCAGCTTGGCTGT TTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAG AAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCA CCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTG TGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAA AGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAAC T pT181 GAATTCTAAAGATCTTTGACAGCTAGCTCAGTCCTAGGTATAATACTAGTAACA attenuator AAATAAAAAGGAGTCGCTCACGCCCTGACCAAAGTTTGTGAACGACATCATTC (EcoRIAAAGAAAAAAACACTGAGTTGTTTTTATAATCTTGTATATTTAGATATTAAACGA J23119TATTTAAATATACATAAAGATATATATTTGGGTGAGCGATTCCTTAAACGAAATT attenuatorGAGATTAAGGAGTCGCTCTTTTTTATGTATAAAAACAATCATGCAAATCATTCA SFGFPAATCATTTGGAAAATCACGATTTAGACAATTTTTCTAAAACCGGCTACTCTAAT TrrnB-PstI) AGCCGGTTGTAAGGATCTAGGAGGAAGGATCTATGAGCAAAGGAGAAGAACT TTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCA CAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAACGGAAAACTC ACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGTGGCCAACACTT GTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACAT GAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAA CGCACTATATCTTTCAAAGATGACGGGACCTACAAGACGCGTGCTGAAGTCAA GTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTATTGATTTTAA AGAAGATGGAAACATTCTTGGACACAAACTCGAGTACAACTTTAACTCACACA ATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAA ATTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACA AAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGT CGACACAATCTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTC CTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTA
2
pT181 antisense (EcoRIJ23119antisenseTrrnB-PstI)
t500 PLlac0-1 Monomeric red fluorescent protein (mRFP)
CAAATAAGGATCTGAAGCTTGGGCCCGAACAAAAACTCATCTCAGAAGAGGAT CTGAATAGCGCCGTCGACCATCATCATCATCATCATTGAGTTTAAACGGTCTC CAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAA ATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGC GCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGC GCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCA TCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTT GTTTGTCGGTGAACTGGATCCTTACTCGAGTCTAGACTGCAG GAATTCTAAAGATCTTTGACAGCTAGCTCAGTCCTAGGTATAATACTAGTATAC AAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAATGATGTCGTTCACAAACT TTGGTCAGGGCGTGAGCGACTCCTTTTTATTTGGATCTGAAGCTTGGGCCCG AACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCAT CATCATCATTGAGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAG AAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAA ACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCC GAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCAT GCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAA GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACTGGATCCTTACTCG AGTCTAGACTGCAG CAAAGCCCGCCGAAAGGCGGGCTTTTTTTT AATTGTGAGCGGATAACAATTGACATTGTGAGCGGATAACAAGATACT TCGGGGGAAATATTCCGAAATGGCAAGTAGCGAAGACGTTATCAAAGAGTTC ATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAAT CGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTG AAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGC AGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGA CTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTATGAACT TCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACGG TGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTC CGGTTATGCAGAAAAAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTA CCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAA GACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAAC CGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTC CCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGT CACTCCACCGGTGCTTAA
Table S2: Attenuator plasmids used in this study. Plasmid sequences can be constructed by replacing the purple region in the pT181 attenuator sequence in Table S1 with the purple region indicated here. White background indicates fusion sequence. Lower case nucleotides indicate mutations made to fusions. All chimeric fusions constructed, with the exception of several non-functional mutants, are reported in this table and in Table S3. Plasmid Figure # Attenuator sequence Name JBL001 TrrnB – CmR – p15A origin AACAAAATAAAAAGGAGTCGCTCACGCCCTGACCAAAGTT 2, 3, 4, TGTGAACGACATCATTCAAAGAAAAAAACACTGAGTTGTT 5, 6, TTTATAATCTTGTATATTTAGATATTAAACGATATTTAAATA S2, S3, TACATAAAGATATATATTTGGGTGAGCGATTCCTTAAACG S5, S6, AAATTGAGATTAAGGAGTCGCTCTTTTTTATGTATAAAAAC S7, S8, AATCATGCAAATCATTCAAATCATTTGGAAAATCACGATTT AGACAATTTTTCTAAAACCGGCTACTCTAATAGCCGGTTG JBL006 TAA pT181 AACAAAATAAAAAGGAGTCGCTCTGTCCCTCGCCAAAGTT 6, S7, JBL007 GCAGAACGACATCATTCAAAGAAAAAAACACTGAGTTGTT pT181.H1 S8
3
JBL009
JBL1037
JBL1126
JBL1016
JBL1048
JBL1815
JBL1017
TTTATAATCTTGTATATTTAGATATTAAACGATATTTAAATA TACATAAAGATATATATTTGGGTGAGCGATTCCTTAAACG AAATTGAGATTAAGGAGTCGCTCTTTTTTATGTATAAAAAC AATCATGCAAATCATTCAAATCATTTGGAAAATCACGATTT AGACAATTTTTCTAAAACCGGCTACTCTAATAGCCGGTTG TAA AACAAAATAAAAAGGAGTCGCTCGTACCCTCTGCAAAGTT AACGAACGACATCATTCAAAGAAAAAAACACTGAGTTGTT TTTATAATCTTGTATATTTAGATATTAAACGATATTTAAATA TACATAAAGATATATATTTGGGTGAGCGATTCCTTAAACG AAATTGAGATTAAGGAGTCGCTCTTTTTTATGTATAAAAAC AATCATGCAAATCATTCAAATCATTTGGAAAATCACGATTT AGACAATTTTTCTAAAACCGGCTACTCTAATAGCCGGTTG TAA AACAAAATAAAAAGGAGTCGCTCACGCTTTGGCGAGTGT GAACGACATCATTCAAAGAAAAAAACACTGAGTTGTTTTT ATAATCTTGTATATTTAGATATTAAACGATATTTAAATATAC ATAAAGATATATATTTGGGTGAGCGATTCCTTAAACGAAA TTGAGATTAAGGAGTCGCTCTTTTTTATGTATAAAAACAAT CATGCAAATCATTCAAATCATTTGGAAAATCACGATTTAGA CAATTTTTCTAAAACCGGCTACTCTAATAGCCGGTTGTAA AACAAAATAAAAAGGAGTCGCTCACGTTCAACTTTGGCGA GTACGATGTGAACGACATCATTCAAAGAAAAAAACACTGA GTTGTTTTTATAATCTTGTATATTTAGATATTAAACGATATT TAAATATACATAAAGATATATATTTGGGTGAGCGATTCCTT AAACGAAATTGAGATTAAGGAGTCGCTCTTTTTTATGTATA AAAACAATCATGCAAATCATTCAAATCATTTGGAAAATCAC GATTTAGACAATTTTTCTAAAACCGGCTACTCTAATAGCC GGTTGTAA AACAAAATAAAAAGGAGTCGCTCACTTTGGCGAGTGAAC GACATCATTCAAAGAAAAAAACACTGAGTTGTTTTTATAAT CTTGTATATTTAGATATTAAACGATATTTAAATATACATAAA GATATATATTTGGGTGAGCGATTCCTTAAACGAAATTGAG ATTAAGGAGTCGCTCTTTTTTATGTATAAAAACAATCATGC AAATCATTCAAATCATTTGGAAAATCACGATTTAGACAATT TTTCTAAAACCGGCTACTCTAATAGCCGGTTGTAA AACAAAATAAAAAGGAGTCGCTCATTCAACTTTGGCGAGT ACGATGAACGACATCATTCAAAGAAAAAAACACTGAGTTG TTTTTATAATCTTGTATATTTAGATATTAAACGATATTTAAA TATACATAAAGATATATATTTGGGTGAGCGATTCCTTAAAC GAAATTGAGATTAAGGAGTCGCTCTTTTTTATGTATAAAAA CAATCATGCAAATCATTCAAATCATTTGGAAAATCACGATT TAGACAATTTTTCTAAAACCGGCTACTCTAATAGCCGGTT GTAA AACAAAATAAAAAGGAGTCGCTCACGCTTGGCGGTGTGA ACGACATCATTCAAAGAAAAAAACACTGAGTTGTTTTTATA ATCTTGTATATTTAGATATTAAACGATATTTAAATATACATA AAGATATATATTTGGGTGAGCGATTCCTTAAACGAAATTG AGATTAAGGAGTCGCTCTTTTTTATGTATAAAAACAATCAT GCAAATCATTCAAATCATTTGGAAAATCACGATTTAGACAA TTTTTCTAAAACCGGCTACTCTAATAGCCGGTTGTAA AACAAAATAAAAAGGAGTCGCTCACGGAACTTGGCGGAA CTGTGAACGACATCATTCAAAGAAAAAAACACTGAGTTGT TTTTATAATCTTGTATATTTAGATATTAAACGATATTTAAAT ATACATAAAGATATATATTTGGGTGAGCGATTCCTTAAAC
6, S7, S8
pT181.YS S2
Fusion 10 3, 5, 6, S5, S7, S8
Fusion 4 S2
Fusion 11 S2
Fusion 12 2
Fusion 1 2 Fusion 2
4
JBL1039
JBL1813
JBL1018
JBL1019
JBL1040
JBL1020
JBL1057
GAAATTGAGATTAAGGAGTCGCTCTTTTTTATGTATAAAAA CAATCATGCAAATCATTCAAATCATTTGGAAAATCACGATT TAGACAATTTTTCTAAAACCGGCTACTCTAATAGCCGGTT GTAA AACAAAATAAAAAGGAGTCGCTCACGCCTCGAACTTGGC GGAACGCAGTGTGAACGACATCATTCAAAGAAAAAAACA CTGAGTTGTTTTTATAATCTTGTATATTTAGATATTAAACG ATATTTAAATATACATAAAGATATATATTTGGGTGAGCGAT TCCTTAAACGAAATTGAGATTAAGGAGTCGCTCTTTTTTAT GTATAAAAACAATCATGCAAATCATTCAAATCATTTGGAAA ATCACGATTTAGACAATTTTTCTAAAACCGGCTACTCTAAT AGCCGGTTGTAA AACAAAATAAAAAGGAGTCGCTCACTTGGCGGTGAACGA CATCATTCAAAGAAAAAAACACTGAGTTGTTTTTATAATCT TGTATATTTAGATATTAAACGATATTTAAATATACATAAAGA TATATATTTGGGTGAGCGATTCCTTAAACGAAATTGAGAT TAAGGAGTCGCTCTTTTTTATGTATAAAAACAATCATGCAA ATCATTCAAATCATTTGGAAAATCACGATTTAGACAATTTT TCTAAAACCGGCTACTCTAATAGCCGGTTGTAA AACAAAATAAAAAGGAGTCGCTCAGAACTTGGCGGAACT GAACGACATCATTCAAAGAAAAAAACACTGAGTTGTTTTT ATAATCTTGTATATTTAGATATTAAACGATATTTAAATATAC ATAAAGATATATATTTGGGTGAGCGATTCCTTAAACGAAA TTGAGATTAAGGAGTCGCTCTTTTTTATGTATAAAAACAAT CATGCAAATCATTCAAATCATTTGGAAAATCACGATTTAGA CAATTTTTCTAAAACCGGCTACTCTAATAGCCGGTTGTAA AACAAAATAAAAAGGAGTCGCTCACCTCGAACTTGGCGG AACGCAGTGAACGACATCATTCAAAGAAAAAAACACTGAG TTGTTTTTATAATCTTGTATATTTAGATATTAAACGATATTT AAATATACATAAAGATATATATTTGGGTGAGCGATTCCTTA AACGAAATTGAGATTAAGGAGTCGCTCTTTTTTATGTATAA AAACAATCATGCAAATCATTCAAATCATTTGGAAAATCACG ATTTAGACAATTTTTCTAAAACCGGCTACTCTAATAGCCG GTTGTAA AACAAAATAAAAAGGAGTCGCTCACGCTTACGAACTTGGC GGAACGACGTGTGAACGACATCATTCAAAGAAAAAAACA CTGAGTTGTTTTTATAATCTTGTATATTTAGATATTAAACG ATATTTAAATATACATAAAGATATATATTTGGGTGAGCGAT TCCTTAAACGAAATTGAGATTAAGGAGTCGCTCTTTTTTAT GTATAAAAACAATCATGCAAATCATTCAAATCATTTGGAAA ATCACGATTTAGACAATTTTTCTAAAACCGGCTACTCTAAT AGCCGGTTGTAA AACAAAATAAAAAGGAGTCGCTCACTTACGAACTTGGCG GAACGACGTGAACGACATCATTCAAAGAAAAAAACACTGA GTTGTTTTTATAATCTTGTATATTTAGATATTAAACGATATT TAAATATACATAAAGATATATATTTGGGTGAGCGATTCCTT AAACGAAATTGAGATTAAGGAGTCGCTCTTTTTTATGTATA AAAACAATCATGCAAATCATTCAAATCATTTGGAAAATCAC GATTTAGACAATTTTTCTAAAACCGGCTACTCTAATAGCC GGTTGTAA AACAAAATAAAAAGGAGTCGCTCACGGATTTTTCGCGAAA CCTGTGAACGACATCATTCAAAGAAAAAAACACTGAGTTG TTTTTATAATCTTGTATATTTAGATATTAAACGATATTTAAA TATACATAAAGATATATATTTGGGTGAGCGATTCCTTAAAC GAAATTGAGATTAAGGAGTCGCTCTTTTTTATGTATAAAAA
2, 5, 6, S5, S7
Fusion 3 S2
Fusion 7 S2
Fusion 8 S2
Fusion 9 3
Fusion 5 3, 6, S7, S8
Fusion 6 S3
Fusion 17
5
JBL1059
JBL1805
JBL1063
JBL1065
JBL1166
JBL1110
JBL1190
CAATCATGCAAATCATTCAAATCATTTGGAAAATCACGATT TAGACAATTTTTCTAAAACCGGCTACTCTAATAGCCGGTT GTAA AACAAAATAAAAAGGAGTCGCTCACGTTGATTTTTCGCGA AACCATTTGTGAACGACATCATTCAAAGAAAAAAACACTG AGTTGTTTTTATAATCTTGTATATTTAGATATTAAACGATAT TTAAATATACATAAAGATATATATTTGGGTGAGCGATTCCT TAAACGAAATTGAGATTAAGGAGTCGCTCTTTTTTATGTAT AAAAACAATCATGCAAATCATTCAAATCATTTGGAAAATCA CGATTTAGACAATTTTTCTAAAACCGGCTACTCTAATAGC CGGTTGTAA AACAAAATAAAAAGGAGTCGCTCACGTCTGATTATTGATT TTTCGCGAAACCATTTAATCATATGTGAACGACATCATTCA AAGAAAAAAACACTGAGTTGTTTTTATAATCTTGTATATTT AGATATTAAACGATATTTAAATATACATAAAGATATATATTT GGGTGAGCGATTCCTTAAACGAAATTGAGATTAAGGAGT CGCTCTTTTTTATGTATAAAAACAATCATGCAAATCATTCA AATCATTTGGAAAATCACGATTTAGACAATTTTTCTAAAAC CGGCTACTCTAATAGCCGGTTGTAA AACAAAATAAAAAGGAGTCGCTCA GATTTTTCGCGAAACC TGAACGACATCATTCAAAGAAAAAAACACTGAGTTGTTTTT ATAATCTTGTATATTTAGATATTAAACGATATTTAAATATAC ATAAAGATATATATTTGGGTGAGCGATTCCTTAAACGAAA TTGAGATTAAGGAGTCGCTCTTTTTTATGTATAAAAACAAT CATGCAAATCATTCAAATCATTTGGAAAATCACGATTTAGA CAATTTTTCTAAAACCGGCTACTCTAATAGCCGGTTGTAA AACAAAATAAAAAGGAGTCGCTCATTGATTTTTCGCGAAA CCATTTGAACGACATCATTCAAAGAAAAAAACACTGAGTT GTTTTTATAATCTTGTATATTTAGATATTAAACGATATTTAA ATATACATAAAGATATATATTTGGGTGAGCGATTCCTTAAA CGAAATTGAGATTAAGGAGTCGCTCTTTTTTATGTATAAAA ACAATCATGCAAATCATTCAAATCATTTGGAAAATCACGAT TTAGACAATTTTTCTAAAACCGGCTACTCTAATAGCCGGT TGTAA AACAAAATAAAAAGGAGTCGCTCATCTGATTATTGATTTTT CGCGAAACCATTTAATCATATGAACGACATCATTCAAAGA AAAAAACACTGAGTTGTTTTTATAATCTTGTATATTTAGAT ATTAAACGATATTTAAATATACATAAAGATATATATTTGGG TGAGCGATTCCTTAAACGAAATTGAGATTAAGGAGTCGCT CTTTTTTATGTATAAAAACAATCATGCAAATCATTCAAATC ATTTGGAAAATCACGATTTAGACAATTTTTCTAAAACCGGC TACTCTAATAGCCGGTTGTAA AACAAAATAAAAAGGAGTCGCTCACCTATGTCTAGTCCAC ATCAGTGAACGACATCATTCAAAGAAAAAAACACTGAGTT GTTTTTATAATCTTGTATATTTAGATATTAAACGATATTTAA ATATACATAAAGATATATATTTGGGTGAGCGATTCCTTAAA CGAAATTGAGATTAAGGAGTCGCTCTTTTTTATGTATAAAA ACAATCATGCAAATCATTCAAATCATTTGGAAAATCACGAT TTAGACAATTTTTCTAAAACCGGCTACTCTAATAGCCGGT TGTAA AACAAAATAAAAAGGAGTCGCTCATCTGATTATTGATTTttG gcGAAACCATTTAATCATATGAACGACATCATTCAAAGAAA AAAACACTGAGTTGTTTTTATAATCTTGTATATTTAGATATT AAACGATATTTAAATATACATAAAGATATATATTTGGGTGA GCGATTCCTTAAACGAAATTGAGATTAAGGAGTCGCTCTT
S3
Fusion 18 S3
Fusion 19 4
Fusion 13 4
Fusion 14 4, 5, 6, S6, S7, S8
Fusion 15 4, 5, 6, S5, S7, S8
Fusion 16 S6 Fusion 15m1
6
JBL1191
JBL1192
JBL1199
JBL1193
JBL1080
JBL1111
JBL1075
TTTTATGTATAAAAACAATCATGCAAATCATTCAAATCATTT GGAAAATCACGATTTAGACAATTTTTCTAAAACCGGCTAC TCTAATAGCCGGTTGTAA AACAAAATAAAAAGGAGTCGCTCATCTGATTATTGATTTtA cgcGAAACCATTTAATCATATGAACGACATCATTCAAAGAA AAAAACACTGAGTTGTTTTTATAATCTTGTATATTTAGATAT TAAACGATATTTAAATATACATAAAGATATATATTTGGGTG AGCGATTCCTTAAACGAAATTGAGATTAAGGAGTCGCTCT TTTTTATGTATAAAAACAATCATGCAAATCATTCAAATCATT TGGAAAATCACGATTTAGACAATTTTTCTAAAACCGGCTA CTCTAATAGCCGGTTGTAA AACAAAATAAAAAGGAGTCGCTCATCTGATTATTGATTTAA GCcGAAACCATTTAATCATATGAACGACATCATTCAAAGAA AAAAACACTGAGTTGTTTTTATAATCTTGTATATTTAGATAT TAAACGATATTTAAATATACATAAAGATATATATTTGGGTG AGCGATTCCTTAAACGAAATTGAGATTAAGGAGTCGCTCT TTTTTATGTATAAAAACAATCATGCAAATCATTCAAATCATT TGGAAAATCACGATTTAGACAATTTTTCTAAAACCGGCTA CTCTAATAGCCGGTTGTAA AACAAAATAAAAAGGAGTCGCTCATCTGATTATTGATTTtA cCcGAAACCATTTAATCATATGAACGACATCATTCAAAGAA AAAAACACTGAGTTGTTTTTATAATCTTGTATATTTAGATAT TAAACGATATTTAAATATACATAAAGATATATATTTGGGTG AGCGATTCCTTAAACGAAATTGAGATTAAGGAGTCGCTCT TTTTTATGTATAAAAACAATCATGCAAATCATTCAAATCATT TGGAAAATCACGATTTAGACAATTTTTCTAAAACCGGCTA CTCTAATAGCCGGTTGTAA AACAAAATAAAAAGGAGTCGCTCATCTGATTATTGATTTcg gggGAAACCATTTAATCATATGAACGACATCATTCAAAGAA AAAAACACTGAGTTGTTTTTATAATCTTGTATATTTAGATAT TAAACGATATTTAAATATACATAAAGATATATATTTGGGTG AGCGATTCCTTAAACGAAATTGAGATTAAGGAGTCGCTCT TTTTTATGTATAAAAACAATCATGCAAATCATTCAAATCATT TGGAAAATCACGATTTAGACAATTTTTCTAAAACCGGCTA CTCTAATAGCCGGTTGTAA AACAAAATAAAAAGGAGTCGCTCACGTTCAtgaTTGGCGtca ACGATGTGAACGACATCATTCAAAGAAAAAAACACTGAGT TGTTTTTATAATCTTGTATATTTAGATATTAAACGATATTTA AATATACATAAAGATATATATTTGGGTGAGCGATTCCTTAA ACGAAATTGAGATTAAGGAGTCGCTCTTTTTTATGTATAAA AACAATCATGCAAATCATTCAAATCATTTGGAAAATCACGA TTTAGACAATTTTTCTAAAACCGGCTACTCTAATAGCCGG TTGTAA AACAAAATAAAAAGGAGTCGCTCACGtgtTTCAACTTTGGC GAGTACGAgcaTGTGAACGACATCATTCAAAGAAAAAAAC ACTGAGTTGTTTTTATAATCTTGTATATTTAGATATTAAAC GATATTTAAATATACATAAAGATATATATTTGGGTGAGCGA TTCCTTAAACGAAATTGAGATTAAGGAGTCGCTCTTTTTTA TGTATAAAAACAATCATGCAAATCATTCAAATCATTTGGAA AATCACGATTTAGACAATTTTTCTAAAACCGGCTACTCTAA TAGCCGGTTGTAA AACAAAATAAAAAGGAGTCGCTCACGTTCAACTTTcaCGA GTACGATGTGAACGACATCATTCAAAGAAAAAAACACTGA GTTGTTTTTATAATCTTGTATATTTAGATATTAAACGATATT TAAATATACATAAAGATATATATTTGGGTGAGCGATTCCTT
S6
Fusion 15m2 S6
Fusion 15m3 S6
Fusion 15m4 5, 6, S6, S7, S8
Fusion 15m5 5, 6, S7, S8
Fusion 4m1 5, 6, S7
Fusion 4m2 S5 Fusion 4m3
7
JBL1113
JBL1163
JBL1165
JBL1164
JBL1150
JBL1146 JBL1145
AAACGAAATTGAGATTAAGGAGTCGCTCTTTTTTATGTATA AAAACAATCATGCAAATCATTCAAATCATTTGGAAAATCAC GATTTAGACAATTTTTCTAAAACCGGCTACTCTAATAGCC GGTTGTAA AACAAAATAAAAAGGAGTCGCTCACGagCAACTTTGGCGA GTACttTGTGAACGACATCATTCAAAGAAAAAAACACTGAG TTGTTTTTATAATCTTGTATATTTAGATATTAAACGATATTT AAATATACATAAAGATATATATTTGGGTGAGCGATTCCTTA AACGAAATTGAGATTAAGGAGTCGCTCTTTTTTATGTATAA AAACAATCATGCAAATCATTCAAATCATTTGGAAAATCACG ATTTAGACAATTTTTCTAAAACCGGCTACTCTAATAGCCG GTTGTAA AACAAAATAAAAAGGAGTCGCTCACGCCTCGAAgTTGGC GcAACGCAGTGTGAACGACATCATTCAAAGAAAAAAACAC TGAGTTGTTTTTATAATCTTGTATATTTAGATATTAAACGAT ATTTAAATATACATAAAGATATATATTTGGGTGAGCGATTC CTTAAACGAAATTGAGATTAAGGAGTCGCTCTTTTTTATGT ATAAAAACAATCATGCAAATCATTCAAATCATTTGGAAAAT CACGATTTAGACAATTTTTCTAAAACCGGCTACTCTAATAG CCGGTTGTAA AACAAAATAAAAAGGAGTCGCTCACGCacCGAACTTGGCG GAACGtcGTGTGAACGACATCATTCAAAGAAAAAAACACT GAGTTGTTTTTATAATCTTGTATATTTAGATATTAAACGATA TTTAAATATACATAAAGATATATATTTGGGTGAGCGATTCC TTAAACGAAATTGAGATTAAGGAGTCGCTCTTTTTTATGTA TAAAAACAATCATGCAAATCATTCAAATCATTTGGAAAATC ACGATTTAGACAATTTTTCTAAAACCGGCTACTCTAATAGC CGGTTGTAA AACAAAATAAAAAGGAGTCGCTCACGCCTgcAACTTGGCG GAAgcCAGTGTGAACGACATCATTCAAAGAAAAAAACACT GAGTTGTTTTTATAATCTTGTATATTTAGATATTAAACGATA TTTAAATATACATAAAGATATATATTTGGGTGAGCGATTCC TTAAACGAAATTGAGATTAAGGAGTCGCTCTTTTTTATGTA TAAAAACAATCATGCAAATCATTCAAATCATTTGGAAAATC ACGATTTAGACAATTTTTCTAAAACCGGCTACTCTAATAGC CGGTTGTAA AACAAAATAAAAAGGAGTCGCTCACCTATGTCTgaTCCAC ATCAGTGAACGACATCATTCAAAGAAAAAAACACTGAGTT GTTTTTATAATCTTGTATATTTAGATATTAAACGATATTTAA ATATACATAAAGATATATATTTGGGTGAGCGATTCCTTAAA CGAAATTGAGATTAAGGAGTCGCTCTTTTTTATGTATAAAA ACAATCATGCAAATCATTCAAATCATTTGGAAAATCACGAT TTAGACAATTTTTCTAAAACCGGCTACTCTAATAGCCGGT TGTAA AACAAAATAAAAAGGAGTCGCTCACCTATGTCTtGTCCAC ATCAGTGAACGACATCATTCAAAGAAAAAAACACTGAGTT GTTTTTATAATCTTGTATATTTAGATATTAAACGATATTTAA ATATACATAAAGATATATATTTGGGTGAGCGATTCCTTAAA CGAAATTGAGATTAAGGAGTCGCTCTTTTTTATGTATAAAA ACAATCATGCAAATCATTCAAATCATTTGGAAAATCACGAT TTAGACAATTTTTCTAAAACCGGCTACTCTAATAGCCGGT TGTAA AACAAAATAAAAAGGAGTCGCTCACCTATGTCTAaTCCAC ATCAGTGAACGACATCATTCAAAGAAAAAAACACTGAGTT GTTTTTATAATCTTGTATATTTAGATATTAAACGATATTTAA
S5
Fusion 4m4 5, 6, S7, S8
Fusion 3m1 5, 6, S7, S8
Fusion 3m2 S5
Fusion 3m3 5, 6, S7
Fusion 16m1 S5
Fusion 16m2 S5 Fusion 16m3
8
ATATACATAAAGATATATATTTGGGTGAGCGATTCCTTAAA CGAAATTGAGATTAAGGAGTCGCTCTTTTTTATGTATAAAA ACAATCATGCAAATCATTCAAATCATTTGGAAAATCACGAT TTAGACAATTTTTCTAAAACCGGCTACTCTAATAGCCGGT TGTAA Table S3: Antisense plasmids used in this study. Plasmid sequences can be constructed by replacing the blue region in the pT181 antisense sequence in Table S1 with the blue region indicated here. White background indicates fusion sequence. Lower case nucleotides indicate mutations made to fusions. All chimeric fusions constructed, with the exception of several non-functional mutants, are reported in this table and in Table S3. Plasmid Figure # Antisense sequence Name No antisense JBL002 J23119 – TrrnB – AmpR – ColE1 origin control 2, 3, 4, 5, 6, ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA S2, S3, TGATGTCGTTCACAAACTTTGGTCAGGGCGTGAGCGACT S5, S6, JBL004 CCTTTTTATTT – TrrnB pT181 S7, S8, ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA 6, S7, TGATGTCGTTCTGCAACTTTGGCGAGGGACAGAGCGACT S8 JBL008 CCTTTTTATTT – TrrnB pT181.H1 ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA 6, S7, TGATGTCGTTCGTTAACTTTGCAGAGGGTACGAGCGACT S8 JBL010 CCTTTTTATTT – TrrnB pT181.YS ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA S2 TGATGTCGTTCACACTCGCCAAAGCGTGAGCGACTCCTT JBL1049 TTTATTT – TrrnB Fusion 10 ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA 3, 5, 6, TGATGTCGTTCACATCGTACTCGCCAAAGTTGAACGTGAG S5, S7, JBL1033 CGACTCCTTTTTATTT – TrrnB Fusion 4 S8 ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA S2 TGATGTCGTTCACTCGCCAAAGTGAGCGACTCCTTTTTAT JBL1026 TT – TrrnB Fusion 11 ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA S2 TGATGTCGTTCATCGTACTCGCCAAAGTTGAATGAGCGAC JBL1034 TCCTTTTTATTT – TrrnB Fusion 12 ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA 2 TGATGTCGTTCACACCGCCAAGCGTGAGCGACTCCTTTTT JBL1812 ATTT – TrrnB Fusion 1 ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA 2 TGATGTCGTTCACAGTTCCGCCAAGTTCCGTGAGCGACT JBL1027 CCTTTTTATTT – TrrnB Fusion 2 ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA 2, 5, 6, TGATGTCGTTCACACTGCGTTCCGCCAAGTTCGAGGCGT S5, S7 JBL1035 GAGCGACTCCTTTTTATTT – TrrnB Fusion 3 ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA S2 TGATGTCGTTCACCGCCAAGTGAGCGACTCCTTTTTATTT JBL1814 – TrrnB Fusion 7 ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA S2 TGATGTCGTTCAGTTCCGCCAAGTTCTGAGCGACTCCTTT JBL1028 TTATTT – TrrnB Fusion 8
9
JBL1806 JBL1064 JBL1095 JBL1096
ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA TGATGTCGTTCACTGCGTTCCGCCAAGTTCGAGGTGAGC GACTCCTTTTTATTT – TrrnB ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA TGATGTCGTTCACACGTCGTTCCGCCAAGTTCGTAAGCG TGAGCGACTCCTTTTTATTT – TrrnB ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA TGATGTCGTTCACGTCGTTCCGCCAAGTTCGTAAGTGAG CGACTCCTTTTTATTT – TrrnB GCGAAAAATCCGTGAGCGACTCCTTTTTATTT – t500 GCGAAAAATCAACGTGAGCGACTCCTTTTTATTT – t500 GCGAAAAATCAATAATCAGACGTGAGCGACTCCTTTTTAT TT – t500 GCGAAAAATCTGAGCGACTCCTTTTTATTT – TrrnB GCGAAAAATCTGAGCGACTCCTTTTTATTT – t500 GCGAAAAATCAATGAGCGACTCCTTTTTATTT – t500
JBL1170
GCGAAAAATCAATAATCAGATGAGCGACTCCTTTTTATTT – t500
Fusion 15
GACTAGACATAGGTGAGCGACTCCTTTTTATTT – t500 gcCaaAAATCAATAATCAGATGAGCGACTCCTTTTTATTT – t500 gcgTaAAATCAATAATCAGATGAGCGACTCCTTTTTATTT – t500 gGCTTAAATCAATAATCAGATGAGCGACTCCTTTTTATTT – t500 gGgTaAAATCAATAATCAGATGAGCGACTCCTTTTTATTT – t500
Fusion 16 Fusion 15m1 Fusion 15m2 Fusion 15m3 Fusion 15m4 Fusion 15m5
JBL1173
ccccgAAATCAATAATCAGATGAGCGACTCCTTTTTATTT – t500 ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA TGATGTCGTTCACATCGTtgaCGCCAAtcaTGAACGTGAGC GACTCCTTTTTATTT – TrrnB ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA TGATGTCGTTCACAtgcTCGTACTCGCCAAAGTTGAAacaC GTGAGCGACTCCTTTTTATTT – TrrnB ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA TGATGTCGTTCACATCGTACTCGtgAAAGTTGAACGTGAG CGACTCCTTTTTATTT – TrrnB ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA TGATGTCGTTCACAaaGTACTCGtgAAAGTTGctCGTGAGC GACTCCTTTTTATTT – TrrnB ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA TGATGTCGTTCACACTGCGTTgCGCCAAcTTCGAGGCGTG AGCGACTCCTTTTTATTT – TrrnB ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA TGATGTCGTTCACACgaCGTTCCGCCAAGTTCGgtGCGTG AGCGACTCCTTTTTATTT – TrrnB ATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAA TGATGTCGTTCACACTGgcTTCCGCCAAGTTgcAGGCGTG AGCGACTCCTTTTTATTT – TrrnB
JBL1180
GAtcAGACATAGGTGAGCGACTCCTTTTTATTT – t500
JBL1024 JBL1036 JBL1029 JBL1119 JBL1093
JBL1133 JBL1194 JBL1195 JBL1196 JBL1197 JBL1198 JBL1081 JBL1074 JBL1076 JBL1120 JBL1169 JBL1174
S2 Fusion 9 3 Fusion 5 3, 6, S7, S8 Fusion 6 Fusion 17 Fusion 18 Fusion 19 Fusion 13 Fusion 13 Fusion 14
S3 S3 S3 4 4 4 4, 5, 6, S6, S7, S8 4, 5, 6, S5, S7 S6 S6 S6 S6 5, 6, S6, S7, S8 5, 6, S7, S8
Fusion 4m1 5, 6, S7 Fusion 4m2 S5 Fusion 4m3 S5 Fusion 4m4 5, 6, S7, S8 Fusion 3m1 5, 6, S7, S8 Fusion 3m2 S5 Fusion 3m3 Fusion 16m1
5, 6, S7 10
JBL1177
GACaAGACATAGGTGAGCGACTCCTTTTTATTT – t500
JBL1167
CCTATGTCTAaTCTGAGCGACTCCTTTTTATTT – t500
Fusion 16m2 Fusion 16m3
S5 S5
Figure S1. (A) Plasmid architecture for antisense and attenuator plasmids. All antisense plasmids have the ColE1 origin and ampicillin resistance (AmpR). All attenuator plasmids have the p15A origin and chloramphenicol resistance (CmR). The J23119 E. Coli consensus promoter (http://partsregistry.org/Part:BBa_J23119), modified to include a SpeI site right before the start of transcription, was used for all plasmids. TrrnB and t500 are transcriptional terminator sequences. RBS = ribosome binding site; SFGFP = super folder green fluorescent protein coding sequence. See Table S1 for sequence details of these plasmids. (B) Diagram of in vivo gene expression assay with example results. E. coli TG1 cells are transformed with a plasmid containing the attenuator transcriptionally fused to SFGFP, and another plasmid encoding antisense (white bar) or a no antisense control plasmid (green bar).
11
B
Normalized Fluorescence/OD
1.2 Fusion 3 Fusion 3 Fit
1.0 0.8 0.6 0.4 0.2
Fusion3-SFGFP
Fusion 3-mRFP
(-) Antisense
(+) Antisense
1.25
Normalized Fluorescence/OD
A
1.00 0.75 0.50 0.25 0
0 10 -7
10 -6
10 -5
10 -4
10 -3
[IPTG], M
Figure S2. Fusion 3 inducibility and functional modularity. (A) Induction curve using an IPTG inducible PLlac0-1 promoter (40) for Fusion 3. Fluorescence/OD are plotted for different concentrations of IPTG in the growth media (see Materials and Methods above). Data were normalized to the no antisense condition. The dashed line shows a Hill equation fit to the data (n = 3.07). Error bars represent standard deviations of at least 4 independent transformants. (B) Comparison of in vivo expression data using SFGFP (grey bars) and monomeric red fluorescent protein (mRFP) (white bars) as reporters for Fusion 3 attenuation. Data were normalized to the no antisense condition in each case. Error bars represent standard deviations of at least 5 independent transformants.
Fusion 4 Antisense 3'
5'
C G A U G C C G G U A U G C U A G C C A A A U G C U G U U A G C A U A C A G CC
U U U C A A C U U G C A C U C G C U G A G G
5'
Fusion 5 Antisense 3'
C G A U G C C G G U A U G C U G C C G G C C A A A A U G C UG U
GG
C G A G UA C G A A U G U G A A C G A C A U C
Fusion 4
A A A U U
C
A
3'
5'
U U C
G C U G G G
Fusion 6 Antisense 3'
C A G C G A G U G A A U G C U U
G U C G U U C A C G
5'
U C G U U C G C A A C CG
GG
G C C G C A C U
5'
C G G
C G
A A A A
A
C G U G U G A A C G A C A U C
A U U
C
A
3'
Fusion 5
5'
U U C
GG
G C C A C U G C U G G G
C G G
C G
A A A
C G U G A A C G A C A U C
3'
Fusion 6
Figure S3. Predicted structures for fusions in Figure 3. Predicted MFE structures of the first hairpin of the chimeric attenuator and antisense for Fusions 4, 5 and 6. Purple and green colored bases represent sequence from the parent translational regulator (see Figure 3 for color code). Bases in black represent the wild type pT181 sequence.
12
Fluorescence/OD
(-) Antisense
(+) Antisense
80,000 60,000
36%
40,000
24%
20,000
27%
84%
0
pT181
33% 72%
88% Fusion 7
Fusion 8
Fusion 9
Fusion 10
Fusion 11
Fusion 12
Figure S4. Testing the interior-loop design principle for loop-loop chimeric attenuators. Data complementary to Figures 2 and 3. Fusions 7, 8 and 9 were engineered using the pT181 position at A24 (Figure 3A) for the three fusion sequences of TransSysM (M1, M2 and M3 – see Figure 2). Fusions 10 and 11 were engineered using a combination of the top loop of the TransSysR hairpin and pT181 positions G26 and A24, respectively. Fusion 12 was engineered using a combination of the TransSysR fusion region in Figure 3 and pT181 position A24. Average in vivo fluorescence from cells with (grey) or without (white) cognate antisense RNA. Error bars represent the standard deviation of at least 5 independent transformations.
I2
I3
TransSys I
60,000
26 24
CC A A G A U A C G C U C U G U C G U A C G U A C A G C C G U A G C A A G U G C
Fluorescence/OD
I1
CG U C U G C G U A U A U A C A G C A U U U U A U U G U A A U G C A U U C A U G U G U A U G C A U A U C G A U U A G C U A G C U G
(-)
(+) Antisense
40,000
20,000
0 pT181 Fus 17 Fus 18 Fus 19 t500 t500 t500
pT181
Figure S5. Chimeric attenuators based on TransSysI and pT181 position G26. Data complementary to Figure 4. Average in vivo fluorescence from cells with (black) and without (white) plasmids encoding cognate antisense RNA for each fusion. The t500 terminator was used for all chimeric antisense plasmids. Error bars represent the standard deviation of at least 5 independent transformations.
13
A
B Energy: -23.7 kcal/mol
Energy: -140.7 kcal/mol T C G A G C C G T T C T C T G T G T T C A A G A G C T T T G 120 A 140
TrrnB G C A A C C G T 280
A
200
G T A G G C G C
A G A C
C G
A
T G
T C A A G T
290
A
G
260
C C
T
G G
240
C T C G C A C G G C T A C C C G T C C G T T A G G G G G T C
G 150 190
A
G
C G T
G
A C G A T A G C T A T
A C
A
220 G G C G G T C T 210 C C G C G T C A G G T T C A A A G A C A A 330 T T C T G T T C
A 320
170
T
230
T
T C T G A
A 180
A
A
A G
A A
C
A G
160
A G
300 C T A C C C A G G G A
G C G T
A T G A G
A G A A G C C G G C G T A C G T A G C A T T A
270 A
A
C G T G T A
T T A G C A T C
T C
T C
G T A A G C A T A T C A
30
T G
130
G T
T
T
110
A
A
C
100
C
G C C C
T
G
90
A A
80 G
A A
350
360 G A
C A
T
G
A
G A
C
A
T
C
C
T
G
G
G
G
A
G
A
C
A
C
G
T
C
T
A
T
A
C
A
G
A
T
70
T
T
T
T
A
GA CT T
380
A
C
A
A 340
T
A
390
A
60
t500
A
A T G C C G C G
50
A A
C
T
50 400
A
G C
G
A G
T
C T C A A T A T A T C G
G
370
A
C
G
G A
C
T A T T T T T G T G A CC T T C 20
G C C G C C C G
A
C
G
G
A
A
A
A 10
A
A
10 C T
A
A
A G G C G G G C T T
A A
T
G
G T G C G T T G T A C G G A C
40
A
60 T A
T T
T
T
G
C
G
A
C
T G
C
G
A
A
A
A 10
A
G T G C G T T G T A C G G A C
G A G
G A C
20
T C T
G A
30 A T G C G T C T T T T A T 40T T
C
20
T C T 30 A T G C G T C T T T T A T 40T T
Figure S6. Linear antisense folding of Fusion 13 with TrrnB and t500 terminators. Predicted minimum free energy (MFE) structure of Fusion 13 antisense with (A) TrrnB and (B) t500; red box highlights the antisense sequence which is zoomed in an inset in (A).
(-) Antisense
Fluorescence/OD
80,000
32%
60,000 28%
25%
31%
40,000 20,000
(+) Antisense
83%
77%
57%
47%
77%
0
pT181
Fusion 4
Fusion 4m3 Fusion 4m4 Fusion 3
Fusion 3m3 Fusion 16 Fusion 16m2 Fusion 16m3
Figure S7. Representative set of chimera mutation failures. Data complementary to Figure 5. Average in vivo fluorescence from cells with (grey) and without (white) plasmids encoding cognate antisense RNA for each fusion. Error bars represent the standard deviation of at least 5 independent transformations.
14
% 100
B
pT181
Mutation - sequence Fusion 15
Fusion 15
m1
Attenuator
m2 m3
80
m1
60
m2 40
m3
m4 m4
m5
20
TransSys I
C
(-) Antisense
(+) pT181
m5
m4
m3
≤0
m2
m1
Fusion 15
m5 pT181
CG U C U G C G U A U A U A C A G C A U U U U A U U G U A A U G C A U U C A U G U G U A U G C A U A U C G A U U A G C U A G C U G
Attenuation
A
Antisense
(+) Fusion 15
(+) m1
(+) m2
(+) m3
(+) m4
(+) m5
Fluorescence/OD
60,000
40,000
20,000
0 pT181
Fusion 15
Fusion 15m1
Fusion 15m2
Fusion 15m3
Fusion 15m4
Fusion 15m5
Attenuator
Figure S8. Mutations to Fusion 15. (A) Five previously reported mutations to the IS10 translational regulator (7) were applied to chimeric attenuator Fusion 15. Mutations were made by substituting the highlighted yellow sequence with sequences listed to the right. (B) 7x7 orthogonality matrix for the pT181 attenuator, Fusion 15, and the 5 mutants. The matrix shows all possible combinations of (antisense, attenuator) for the 7 attenuators tested. Attenuation % is represented by a color scale in which 100% is blue and less than or equal to 0% is white. (C) In vivo expression data used to calculate the matrix in part (B). White bars represent the average fluorescence/OD of cells without antisense and colored bars of cells with antisense according to the legend. Error bars represent standard deviations of at least 4 independent transformants.
15
0
Antisense
Antisense
80,000
40,000
20,000
0
40,000
20,000
Fusion 15
Fusion 4m2
Fusion 16m1
Fusion 15m5 Fusion 6
Fusion 16m1
Fusion 15m5
Fusion 6
Fusion 3m2
60,000
Fusion 3m2
Antisense
Fusion 3m1
Fusion 6 attenuator
Fusion 3m1
Fusion 4m2
Fusion 15m5 attenuator
Fusion 4m1
Antisense Fusion 6
Fusion 15m5
Fusion 16m1
Fusion 3m2
Fusion 3m1
Fusion 4m2
Fusion 4m1
Fusion 15
Fusion 4
Antisense
Fusion 15
0
Antisense
60,000
40,000
20,000
Fusion 3m1
Fusion 4m2
Fusion 4m1
Fusion 15
Fusion 4
Fusion 3
Fusion 16
pT181.YS
Fusion 16m1 Fusion 15m5 Fusion 6
Fusion 16m1 Fusion 15m5 Fusion 6
Fusion 3m2
Fusion 4m1 attenuator
Fusion 3m2
Fusion 3m1
Fusion 4m2
Fusion 4m1
Fusion 15
Fusion 4
Fusion 3
Antisense
Fusion 4m1
80,000
Fusion 3
80,000
Fusion 16
Antisense
Fusion 4
20,000
Fusion 6
Fusion 15m5
Fusion 16m1
Fusion 3m2
Fusion 3m1
Fusion 4m2
Fusion 4m1
Fusion 15
Fusion 4
Fusion 3
Fusion 16
pT181.YS
pT181.H1
Antisense
Fusion 4
40,000
pT181.YS
80,000
Fusion 16
80,000
Fusion 3
Fusion 3m2 attenuator pT181.H1
Fusion 3 attenuator
Fusion 3
0
pT181.YS
20,000
pT181
80,000
Fusion 16
40,000
pT181.YS
Fusion 4m2 attenuator No Antisense
Fusion 3 Fusion 4
Fusion 6
Fusion 15m5
Fusion 16m1
Fusion 3m2
Fusion 3m1
Fusion 4m2
Fusion 4m1
Fusion 15
Fusion 4
Fusion 3
Fusion 16
pT181.YS
pT181.H1
pT181
No Antisense
Fusion 6
Fusion 15m5
Fusion 16m1
Fusion 3m2
Fusion 3m1
Fusion 4m2
Fusion 4m1
Fusion 15
Fluorescence/OD 80,000
Fusion 16
0
pT181.YS
20,000
pT181.H1
Fusion 15 attenuator
pT181.H1
40,000 0
pT181.H1
20,000
pT181.H1
40,000
pT181
0
No Antisense
20,000
pT181
40,000
No Antisense
60,000
Fluorescence/OD
pT181.YS attenuator
pT181
60,000
Fluorescence/OD
Fusion 6
Fusion 15m5
Fusion 16m1
Fusion 3m2
Fusion 3m1
Fusion 4m2
Fusion 4m1
Fusion 15
Fusion 16
0
No Antisense
60,000
Fluorescence/OD
Fusion 6
Fusion 15m5
Fusion 16m1
Fusion 3m2
Fusion 3m1
Fusion 4m2
Fusion 4m1
Fusion 15
Fusion 4
Fusion 4
Fusion 3
Fusion 16
20,000
pT181
60,000
Fluorescence/OD
Fusion 6
Fusion 15m5
Fusion 16m1
Fusion 3m2
Fusion 3m1
Fusion 4m2
Fusion 4m1
Fusion 15
Fusion 4
Fusion 3
Fusion 16
pT181.YS
pT181.H1
pT181
No Antisense
40,000
No Antisense
60,000
Fluorescence/OD
Fusion 6
Fusion 15m5
Fusion 16m1
Fusion 3m2
Fusion 3m1
Fusion 4m2
Fusion 4m1
Fusion 15
Fusion 4
Fusion 3
pT181.YS
pT181.H1
pT181
No Antisense
Fluorescence/OD 60,000
pT181
60,000
Fluorescence/OD
Fusion 6
Fusion 15m5
Fusion 16m1
Fusion 3m2
Fusion 3m1
Fusion 4m2
Fusion 4m1
Fusion 15
Fusion 4
Fusion 3
pT181.YS
pT181.H1
pT181
No Antisense
Fluorescence/OD
pT181 attenuator
No Antisense
Fusion 6
Fusion 15m5
Fusion 16m1
Fusion 3m2
Fusion 3m1
Fusion 4m2
Fusion 4m1
Fusion 15
Fusion 4
80,000
Fusion 3
80,000
Fusion 3
80,000
Fusion 16
pT181.YS
pT181.H1
80,000
Fusion 16
pT181.YS
pT181.H1
pT181
No Antisense
80,000
Fusion 16
pT181.YS
pT181.H1
pT181
Fluorescence/OD 80,000
Fusion 16
pT181.YS
0
pT181.H1
0
No Antisense
0
pT181
Fluorescence/OD
0
No Antisense
Fluorescence/OD 0
pT181
Fluorescence/OD 0
No Antisense
Fluorescence/OD
80,000
pT181.H1 attenuator
60,000
40,000
20,000
Fusion 16 attenuator
Antisense
60,000
40,000
20,000
Antisense
Fusion 4 attenuator
60,000
40,000
20,000
Antisense
60,000
Fusion 3m1 attenuator
40,000
20,000
Antisense
Fusion 16m1 attenuator
60,000
40,000
20,000
Antisense
16
Figure S9. In vivo expression data used to calculate the 14x14 attenuation matrix found in Figure 6A. White bars represent the average fluorescence/OD of cells without antisense and colored bars of cells with antisense according to label on x-axis. Each plot represents a row of the 14x14 matrix in Figure 6A – i.e. attenuation for a single chimeric attenuator with different combinations of antisense RNA indicated on the x-axis. Error bars represent standard deviations of at least 5 independent transformants.
Normalized Fluorescence
1000
500
Fusion 4m1
1.25
Bulk Fl.
1.00
Flow Cyt.
Background (-) Antisense (+) Antisense
0.75 0.50 0.25 0
(-)
(+) Antisense
1000
500
Bulk Fl.
1.00
Flow Cyt.
0.75 0.50
500
0.25 0
(-)
Bulk Fl.
1.00
Flow Cyt.
0.75 0.50 0.25 0
(-)
(+) Antisense
1000 Normalized Fluorescence
500
pT181.YS
1.25
Bulk Fl.
1.00
Flow Cyt.
0.75
Counts
Normalized Fluorescence
0.50 0.25 0
(-)
500
(+) Antisense
1000
Fusion 15m5
1.25
Bulk Fl.
1.00
Flow Cyt.
0.75 0.50 0.25 0
(-)
(+) Antisense
500
Normalized Fluorescence
Normalized Fluorescence
1000
pT181.H1
1.25
Bulk Fl.
1.00
Flow Cyt.
0.75 0.50
500
0.25 0
(-)
(+) Antisense
1000
Fusion 3m2
1.25
Bulk Fl.
1.00
Flow Cyt.
0.75 0.50 0.25 0
(-)
(+) Antisense
Normalized Fluorescence
1000
500
1.25
Bulk Fl.
1.00
Normalized Fluorescence
Counts
Fusion 6
1.25
(+) Antisense
1000
pT181
Flow Cyt.
0.75 0.50
500
0.25 0
(-)
(+) Antisense
0
1.25
Bulk Fl.
1.00
Fusion 3m1
Flow Cyt.
0.75 0.50 0.25 0
(-)
(+) Antisense
0 1
Normalized Fluorescence
Normalized Fluorescence
1000
Fusion 4
1.25
6
36 219 1329 Arbitrary Fluorescence Units
8058
1
6
36 219 1329 Arbitrary Fluorescence Units
8058
17
Figure S10. Representative flow cytometry data. Representative histograms for the eight attenuators found in the final orthogonality matrix and the pT181 attenuator. Red traces represent cells without (-) antisense, black traces represent cells with (+) cognate antisense, and grey traces represent the background fluorescence of cells not expressing SFGFP. Histograms are plotted on a log-scale in terms of relative intensity, but were calculated in relative channel number as described in the SI Materials and Methods above. Inset plots are a comparison between average bulk fluorescence (white bars) and mean fluorescence calculated from flow cytometry (grey bars) for the (-) and (+) cognate antisense conditions. Average fluorescence was normalized by the fluorescence observed without antisense for each attenuator. Error bars represent standard deviations of at least 3 independent transformants. References 7. Mutalik,V.K., Qi,L., Guimaraes,J.C., Lucks,J.B. and Arkin,A.P. (2012) Rationally designed families of orthogonal RNA regulators of translation. Nat Chem Biol, 8, 447–454. 40. Lutz,R. and Bujard,H. (1997) Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res, 25, 1203– 1210.
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