Transcriptional Interference in Convergent Promoters as a Means for Tunable Gene

Transcription

1 Supporting material for: Transcriptional Interference in Convergent Promoters as a Means for Tunable Gene Expression Antoni E. Bordoy, Usha S. Varanasi, Colleen M. Courtney, and Anushree Chatterjee Department of Chemical and Biological Engineering, University of Colorado Boulder, 3415 Colorado Avenue, UCB 596, Boulder, Colorado 80303, United States. Contents Supporting Text 1: Overlapping DNA generation and plasmid assembly 2 Supporting Text 2: Protein expression in different bacterial strains.6 Supporting Text 3: Statistical and Hill equation analysis.7 Table S1: Primer and oligonucleotide sequences..8 Table S2: Sequences of plasmid constructs.10 Table S3: Parameters resulting of the Hill equation fit to the experimental data for GFP expression 12 Figure S1: Plasmid construct design Figure S2: Absence of RNA interaction for complementary transcripts with an overlapping DNA length of 145 bp...14 Figure S3: Effect of P LLac activity on GFP and mcherry expression...15 Figure S4: Effect of 5 UTR length on protein expression...16 References 17 1

2 Supporting Text 1: Overlapping DNA generation and plasmid assembly Convergent constructs: The overlapping DNA sequences were designed based on two naturally occurring systems demonstrating RNA interaction between sense and antisense transcripts. These systems include: hoksok 1,2 RNA and srnb-srnc 2 RNA, and which are based on the toxin-antitoxin systems found in E.coli. We renamed the three partial sequences used as S1, S2 and S3 (Table S1). The sequence of the bacterial his pause was also used 3 in order to show how pause sequences might play an important role during antisense transcription. Oligonucleotides were ordered from Life Technologies (Thermo Fischer). Different overlapping DNA sequences separating promoters P LTet and P LLac were synthesized making sure that the desired product was generated at the end of each required step by running the product on a 1% agarose electrophoresis gel. Resulting plasmids were transformed into chemically competent E. coli strain DH5αZ1 (Expressys), unless otherwise stated. Cells were made competent using Z-Competent E. Coli Buffer set (Zymo Research Corporation) as per the provided supplier protocol. puv4 (not used in this study) was obtained by inserting GFP (egfp) amplified from pakgfp1 (Addgene plasmid #14076) using primers SalI-RBS2-gfp (f), which includes a designed ribosome binding site (RBS2, Table S1), and gfp_bamhi (r) (all primers used are listed in Table S1) between SalI and BamH1 sites of pze21mcs (Expressys) and later inserting KpnI-S1-S2-SalI fragment into KpnI and SalI restriction sites of that plasmid. KpnI-S1-S2-SalI fragment was generated by mixing sense and overlapping antisense primers as follows: Separate mixtures of oligomers S1 and S2r-S1r, and S2 and S2r-SalI were subjected to denaturation at 95ºC for 2 min, annealing by cooling at 1ºC/min until 30ºC, and extension by adding dntps and Taq polymerase at 72ºC for 30 min in a Bio-Rad thermal cycler. The products were cleaned up using Qiagen PCR purification kit. Eluted DNA was used as template for PCR. Cleaned PCR products served as starting material for next step whereby the obtained products were mixed and the resulting product was amplified using primers KpnI-S1 (f) and S2r-SalI (r). Similarly, the mcherry coding sequence along with its transcriptional termination sequence was amplified from 2

3 pfpv-mcherry (Addgene plasmid #20956) with forward primer XhoI-RBS2-mC (f), downstream of the XhoI site, and reverse primer mc_aatii (r) and cloned into the resulting product of the previous step. puv145 was generated by inserting KpnI-S1-S2-S3-P LLac -SalI fragment into KpnI and SalI sites of puv4. In this case, oligomers were mixed as follows: Set 1 (S1 and S2r-S1r), set 2 (S2 and S3r-S2r) and set 3 (S3 and P LLac -S3r) followed a process of denaturation at 95ºC for 2 min, annealing by cooling at 1ºC/min until 30ºC, and extension by adding dntps and Taq polymerase at 72ºC for 30 min in a Bio-Rad thermal cycler. The products were cleaned up using Qiagen PCR purification kit. Eluted DNA was used as template for PCR. Cleaned PCR products served as starting material for next SOE-PCR step whereby separate mixtures of set 1 and set 2, and set 2 and set 3 were stitched by repeating the procedure to give products, set 4 and set 5 respectively. The process was repeated by mixing set 4 and set 5, obtaining set 6. Separately, set 7 was created by mixing P LLac rev sense oligomer with p2-sali (r). In the fourth step, the SOE-PCR product of mixing set 6 and set 7 containing S1-S2-S3-P LLac was amplified with primers containing restriction sites Kpn1 (KpnI-S1 (f)) and SalI (P LLac -SalI (r)). puv145 was used as a template to amplify XhoI-S1-S2-S3-P Llac -SalI product using AARC S1 Xho_F and P LLac -SalI (r) primers which was then digested with XhoI and SalI and inserted in the product of digestion of puv145 with XhoI (XhoI and SalI are isocaudomers, i.e. they share the four nucleotide sequence of their sticky ends) in order to create pae_l145. Correct insert orientation was certified by sequencing (GENEWIZ). puv174 was created by inserting KpnI-S1-His-S2-S3-P LLac -SalI fragment into KpnI and SalI sites of puv4. In this case, analogous to creation of KpnI-S1-S2-S3-P LLac -SalI fragment, the SOE-PCR process started with separated mixtures of primers S1 with Hisr-S1r and His with S2r-Hisr. Then the resulting products were mixed with set 2 and set 3 and continued as previously described in order to create the product S1-His-S2-S3-P LLac. Which was later amplified using primers containing restriction sites Kpn1 (KpnI-S1 (f)) and SalI (P LLac -SalI (r)). 3

4 puv235 was generated by inserting the XhoI-SalI fragment obtained from pae_l145 (XhoI-S1- S2-S3-P Llac -SalI) into SalI site of puv4. Therefore, to the original S1 and S2 present in the vector puv4, S1-S2-S3-P Llac fragment was added, resulting in an overlapping DNA sequence, i.e. sequence separating promoters P LTet and P Llac, containing S1-S2-S1-S2-S3 bacterial stem loops. Single promoter constructs: pae_t145, pae_t174 and pae_t235 were constructed by amplifying the KpnI-S1-S2-S3-SalI, KpnI-S1-His-S2-S3-SalI and KpnI-S1-S2-S1-S2-S3-SalI sequences, respectively, using puv145, puv174 and puv235, respectively, as a template with primers KpnI-S1 (f) and S3r-SalI (r) and inserting the corresponding resulting product into the KpnI and SalI site of puv4. Correct plasmid assembly was certified by sequencing (GENEWIZ). Similarly, pae_l174 and pae_l235 were constructed by amplifying the XhoI-S1-His-S2- S3-SalI and XhoI-S1-S2-S1-S2-S3-SalI sequences, using puv174 and puv235, respectively, as a template with primers AARC S1 Xho_F and P LLac -SalI (r) and inserting the corresponding resulting product into the XhoI and SalI site of puv4. Correct insert orientation was certified by checking for mcherry production in presence of IPTG in DH5α Wild Type (DH5α-WT) and checking for correct size fragment insertion using PCR amplification with sequence specific primers AARC S1 Xho_F and P LLac - SalI and running the resulting products on a 1% agarose gel. Construct containing bla gene encoded within overlapping DNA: puvbla was generated by firstly inserting the XhoI-AmpR-SalI fragment obtained by amplifying the open reading frame of the TEM-1 bla gene from pze12mcs (Expressys), encoding for a -lactamase enzyme that confers microorganisms with resistance to -lactam antibiotics such as ampicillin, with primers Amp_F-EcoRI and Amp_R-EcoRI and inserting resulting fragment into the EcoRI site of puv19.2 (not used here), which was created by deleting the overlapping sequence (S1-S2-S3) from puv145 by digesting it with EcoRI followed by religation. 4

5 Dual plasmid system for RNA-RNA interaction experiments: In order to test possible RNA-RNA interaction occurring between the sense and antisense transcripts produced in convergent constructs we designed and built a dual plasmid system where each transcript is produced by a different plasmid, therefore the complementary transcripts are expressed in trans. We replaced the original resistance marker of pae_l145, KanR, for AmpR and the original origin of replication, ColE1, for p15a (Fig. S1). To do so, pae_l145 was double digested with FastDigest enzymes XmaJI and AatII. AmpR fragment with its constitutive promoter was obtained by digesting pze12luc (Expressys) with FastDigest enzymes AatII and SacI, the resulting fragment lacks the AmpR transcriptional terminator lambda t0, which was obtained jointly with p15a origin of replication by digesting pza31luc with SacI and XmaJI. Ligation of the three fragments was performed overnight at 16ºC using T4 DNA ligase (Thermo Fisher). Drop dialysis was performed on 5 μl of the ligation mixture following transformation of the resulting plasmid, named pae_l145_r in DH5α-WT. Correct plasmid assembly was confirmed running the products of PCR amplification of S1-S2-S3-P LLac, AmpR and p15a using primers AARC S1 Xho_F and P LLac -SalI (r), AmpR_res_f and AmpR_res_r, and p15a_r and p15a_f, respectively, on a 1% agarose gel. Single promoter construct pae_l145_r was co-transformed with single promoter construct pae_t145 into C600Zi bacterial strain of E. coli. Transformants were grown on double selection LB plates supplemented with Kanamycin (50 μg/ml) and Ampicillin (50 μg/ml). Presence of both plasmids was also confirmed by PCR amplifying fragments S1-S2-S3-P LLac with primers AARC S1 Xho_F and P LLac -SalI (r) for plasmid pae_l145_r, and fragment P LTet -S1-S2-S3 with primers plteto_f and S3r-SalI (r) for plasmid pae_t145. Further proof was obtained by amplifying corresponding resistance markers and p15a origin of replication with primers AmpR_res_f and AmpR_res_r, and p15a_r and p15a_f, respectively, and running them on a 1% agarose gel in order to check for their correct DNA lengths. 5

6 puv145placmut Construct puv145 was used as a template to amplify fragment DraIII-S2-S3-LacO-SalI with primers DraIII-S2-S3 (f) and SalI-PLacmut (r) which incorporate mutations at the -35 (TTGACA AAAGGC) and -10 (GATACT ACCTGG) regions 4 keeping the LacI binding sites intact. The resulting fragment was later digested with DraIII and SalI FastDigest restrictions enzymes and inserted intro puv145 backbone. Correct mutations were verified by sequencing (GENEWIZ). Supporting Text 2: Protein expression in different bacterial strains. In order to quantify the decrease in GFP gene expression due to RNAP roadblock caused by presence of antisense LacI repressor protein, we transferred each of the convergent constructs (puv145, puv174 and puv235) into E. coli bacterial strain C600Zi (Expressys), which expresses LacI while lacks expression of TetR. To do so, plasmids were extracted from 5 ml overnight cultures of the original E. coli bacterial strain DH5αZ1 in LB supplemented with Kanamycin (50 μg/ml) using GeneJET Plasmid Miniprep Kit (Thermo Fisher) as per manufacturers protocol. Extracted plasmids were later transformed into chemically competent E. coli bacterial strain C600Zi (Expressys) as per the provided supplier protocol. Cells made competent using Z-Competent E. Coli Buffer set (Zymo Research Corporation). Similarly, in order to quantify the effect of the presence of active transcription from an antisense downstream convergent promoter, we transferred each of the convergent constructs (puv145, puv174 and puv235) into E. coli bacterial strain DH5α Wild Type (DH5α-WT), which does not express either LacI or TetR repressor proteins following the previously described plasmid extraction and transformation procedures. In order to quantify the decrease in mcherry gene expression due to RNAP roadblock caused by presence of antisense TetR repressor protein, we transferred the pzs4inttetr plasmid (Expressys) into competent DH5α-WT E. coli already harboring one of the convergent constructs (puv145, puv174 and 6

7 puv235). Successful presence of both plasmids was confirmed by growing transformant colonies in double antibiotic selection LB and agar plates supplemented with Kanamycin (50 μg/ml) and Spectinomycin (50 μg/ml). Additionally, PCR confirmation was conducted running on a 1% agarose gel the products obtained using sequence specific primers AARC S1 Xho_F and P LLac -SalI (r) in order to amplify fragments S1-S2-S3-P LLac, S1-His-S2-S3-P LLac and S1-S2-S1-S2-S3-P LLac, respectively, and primers TetR_F_UV and TetR_R_UV to amplify TetR open reading frame from the plasmids extracted from successful transformants as described previously. Supporting Text 3: Statistical and Hill equation analysis. For each set of data presented, three biological replicates, originating from individual colonies, were measured. One-tail t-test for paired samples for means was conducted and the resulting p-values indicated significant differences if their values were less than For clarification, the FACS data presented in Fig. 5c-f and Fig. 6d-e was analyzed as follows: for each atc condition, significant differences in GFP expression were tested for all possible combinations of two different IPTG levels and depicted as brackets in the figures if found to be significant; similarly, for each IPTG condition, significant differences in mcherry expression were tested for all possible combinations of two different atc levels and also depicted as brackets in the figures if found to be significant. GFP expression switching responses at each level of IPTG were fitted (using Microsoft Excel Solver tool) to a Hill equation: Fluorescence( AU..) y min ( ymax ymin ) [ atc] H H K [ atc] H, where constraints were applied to the values of y min and y max to lie in the range of 80 to 120% of the minimum and maximum, respectively, of the data fitted. Parameters were compared within each construct (at different IPTG levels) and only K and H were also compared between constructs because comparison of y min and y max would not be adequate due to inherent significant protein expression levels due to 5 UTR regulation as showed in Fig. S3. Average, standard deviation values and significant differences can be found in Table S3. 7

8 Table S1: Primer and oligonucleotide sequences. Primer/ Oligomer P LTet P LLac P LLac rev sense S1 S2 S3 His S2r-S1r S3r-S2r P LLac -S3r Hisr-S1r S2r-Hisr KpnI-S1 (f) S2r-SalI (r) Sequence (5 3 ) TCCCTATCAGTGATAGAGAT TGACATCCCTATCAGTGATA GAGATACTGAGCACATCAGC AGGACGCACTGACC CTCGAGAATTGTGAGCGGAT AACAATTGACATTGTGAGCG GATAACAAGATACTGAGCAC ATCAGCAGGACGCACTGACT GAATTC GAATTCgGTCAGTGCGTCCTG CTGATGTGCTCAGTATCTTGT TATCCGCTCACAATGTCAATT GTTATCCGCTCACAATTCTCG AG TAGGGATGCCTCGTGGTcGTT AAcGAAAATTAACTTACTAC GGGC CTTTTCTTTGATGTCCCCATT TTGTGGAGCCCATCAAAAG ACCCCACTTGTTAATCCATTA ACTCGTGAtGTC TCTTTCAGGCGATGTGTGCTG GAAGACATTCAGA ATGGGGACATCAAAGAAAAG GCCCGTAGTAAGTTAATTTT AATGGATTAACAAGTGGGGT CTTTTGATGGGCTCCACAAA AGGACGCACTGACCGAATTC GACATCACGAGTTAATGGAT ACATCGCCTGAAAGAGCCCG TAGTAAGTTAA ACATCAAAGAAAAGATCTGA ATGTCTTCCAG GGTACCGGTACCTAGGGATG CCTCGTGGTCGT GTCGACGTCGACCTTTTGATG GGCTCCACAAAATG Description/References pltet-o, pze21-mcs 5 pllac-o, pze12luc 5 Reverse pllac-o sequence. Mutations indicated in lower case. E. coli strain 3A11 plasmid phn3a11. Stem loop of sok RNA from 10 to 55 bp 1. Mutations indicated in lower case. F plasmid (from E.coli) stable RNA degradation promoter gene, srnb. F plasmid (from E. coli) stem loop of stable RNA degradation promoter gene, srnb, from 54 to 86 bp 2. Mutations indicated in lower case. E. coli his leader pause site operon 3. Reverse complementary 3 end of S1 sequence joined with reverse complementary 5 end of S2 sequence Reverse complementary 3 end of S2 sequence joined with reverse complementary 5 end of S3 sequence Reverse complementary 3 end of S3 sequence joined with reverse complementary 5 end of P LLac sequence Reverse complementary 3 end of S1 sequence joined with reverse complementary 5 end of his sequence Reverse complementary 3 end of his sequence joined with reverse complementary 5 end of S2 sequence Duplicated restriction enzyme KpnI site joined with 5 end of S1 sequence Duplicated restriction enzyme SalI site joined with reverse complementary 3 end of S2 sequence 8

9 S3r-SalI (r) P LLac -SalI (r) SalI-RBS2-gfp (f) gfp_bamhi (r) mc_aatii (r) XhoI-RBS2-mC (f) AARC S1 Xho_F Amp_F-EcoRI Amp_R-EcoRI GTCGACGTCGACGACATCAC GAGTTAATGGATTAA GTCGACGTCGACCTCGAGAA TTGTGAGCGGATAAC GTCGACGTCGACAGaAGGAT GAGTAAAGGAGAAGAACTTT TCACT GGATCCGGATCCCTATTTGTA TAGTTCATCCATGCCAT GACGTCGACGTCGGTGCCGA GGATGACGATGA CTCGAGCTCGAGAGGAGGAG ATATACATATGGTGAGCA CTCCAGCTCCAGTAGGGATG CCTCGTGGTCGTTAA GAATTCGAATTCATGAGTAT TCAACATTTCCGTG GAATTCGAATTCTTACCAAT GCTTAATCAGTGAG Duplicated restriction enzyme SalI site joined with reverse complementary 3 end of S3 sequence Duplicated restriction enzyme SalI site joined with reverse complementary 3 end P LLac sequence Duplicated restriction enzyme SalI site joined with RBS2 (AGaAGG) fused with 5 end of GFP sequence. Mutations indicated in lower case Duplicated restriction enzyme BamHI site joined with reverse complementary 3 end of GFP sequence Duplicated restriction enzyme AatII site joined with reverse complementary 3 end of mcherry sequence Duplicated restriction enzyme XhoI site joined with RBS2 (AGGAGG) fused with 5 end of mcherry sequence Duplicated restriction enzyme XhoI site joined with 5 end S1 sequence Duplicated restriction enzyme EcoRI site joined with 5 end of TEM-1 bla sequence Duplicated restriction enzyme EcoRI site joined with reverse complementary 3 end of TEM-1 bla sequence Sequence complementary to the 3 UTR of TEM-1 bla sequence AmpR_res_F CGAGCTCGTAAACTTGGTCT GA AmpR_res_R GTGAAGACGAAAGGGCCTCG Sequence complementary to upstream fragment of TEM-1 bla promoter p15a_f GGGCCGCGGCAAAGCCGTTT Sequence complementary to the 3 end of TTCCATAGG p15a origin of replication p15a_r GCACTAGTAACAACTTATAT Sequence complementary to upstream CGTA fragment of p15a plteto_f CTCGAGTCCCTATCAGTGAT Sequence containing restriction enzyme XhoI AG site joined with partial sequence of Tet TetR_F_UV TetR_R_UV DraIII-S2-S3 (r) SalI-PLacmut (f) GAATTCATATGTCTAGATTA GATAAAAGTAAAG AAGCTTAAGACCCACTTTCA CATTTAAG CACTTTGTGCACTTTGTGGAG CCCATCAAAAGACCC GTCGACGTCGACCTCGAGAA TTGTGAGCGGATAACAAaaagg cttgtgagcggataacaaacct gggagcacatcagc operator Sequence containing 5 end of TetR open reading frame Sequence complementary to 3 end of TetR open reading frame Duplicated restriction enzyme DraIII site, which is present in S2 sequence, joined with 13 bp of 3 end of S2 sequence Duplicated restriction enzyme SalI site joined with complementary sequence of 3 end of reverse P LLac promoter. Mutations at the -10 and -35 regions are indicated in lower case. 9

10 Table S2: Sequences of plasmid constructs Construct name: Sequence (5 3 ) of the sense strand of the variable region shown in Fig. S1. Promoter P LTet. Promoter P LLac. Stem loop S1. Stem loop S2. Stem loop S3. his pause. TEM-1 bla orf. puv4: ATGTATATCTCCTCCTCTCGAGTCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAG AGATACTGAGCACATCAGCAGGACGCACTGACCGAATTCATTAAAGAGGAGAAAGGTACCT AGGGATGCCTCGTGGTCGTTAACGAAAATTAACTTACTACGGGCCTTTTCTTTGATGTCCCCA TTTTGTGGAGCCCATCAAAAGGTCGACAGAAGG puv145: ATGTATATCTCCTCCTCTCGAGTCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAG AGATACTGAGCACATCAGCAGGACGCACTGACCGAATTCATTAAAGAGGAGAAAGGTACCG GTACCTAGGGATGCCTCGTGGTCGTTAACGAAAATTAACTTACTACGGGACTTTTCTTTGATG TCCCCACTTTGTGGAGCCCATCAAAAGACCCCACTTGTTAATCCATTAACTCGTGATGTTGAA TTCAGTCAGTGCGTCCTGCTGATGTGCTCAGTATCTTGTTATCCGCTCACAATGTCAATTGTT ATCCGCTCACAATTCTCGAGGTCGACAGAAGG puv174: ATGTATATCTCCTCCTCTCGAGTCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAG AGATACTGAGCACATCAGCAGGACGCACTGACCGAATTCATTAAAGAGGAGAAAGGTACCT AGGGATGCCTCGTGGTCGTTAACGAAAATTAACTTACTACGGGCTCTTTCAGGCGATGTGTG CTGGAAGACATTCAGATCTTTTCTTTGATGTCCCCATTTTGTGGAGCCCATCAAAAGACCCCA CTTGTTAATCCATTAACTCGTGATGTCGAATTCGGTCAGTGCGTCCTGCTGATGTGCTCAGTA TCTTGTTATCCGCTCACAATGTCAATTGTTATCCGCTCACAATTCTCGAGGTCGACAGAAGG puv235: ATGTATATCTCCTCCTCTCGAGTCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAG AGATACTGAGCACATCAGCAGGACGCACTGACCGAATTCATTAAAGAGGAGAAAGGTACCT AGGGATGCCTCGTGGTCGTTAACGAAAATTAACTTACTACGGGCCTTTTCTTTGATGTCCCCA TTTTGTGGAGCCCATCAAAAGGTCGAGTAGGGATGCCTCGTGGTCGTTAACGAAAATTAACT TACTACGGGACTTTTCTTTGATGTCCCCACTTTGTGGAGCCCATCAAAAGACCCCACTTGTTA ATCCATTAACTCGTGATGTTGAATTCAGTCAGTGCGTCCTGCTGATGTGCTCAGTATCTTGTT ATCCGCTCACAATGTCAATTGTTATCCGCTCACAATTCTCGACAGAAGG pae_t145: ATGTATATCTCCTCCTCTCGAGTCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAG AGATACTGAGCACATCAGCAGGACGCACTGACCGAATTCATTAAAGAGGAGAAAGGTACCG GTACCTAGGGATGCCTCGTGGTCGTTAACGAAAATTAACTTACTACGGGACTTTTCTTTGATG TCCCCACTTTGTGGAGCCCATCAAAAGACCCCACTTGTTAATCCATTAACTCGTGATGTGTCG ACAGAAGG pae_t174: ATGTATATCTCCTCCTCTCGAGTCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAG AGATACTGAGCACATCAGCAGGACGCACTGACCGAATTCATTAAAGAGGAGAAAGGTACCT AGGGATGCCTCGTGGTCGTTAACGAAAATTAACTTACTACGGGCTCTTTCAGGCGATGTGTG CTGGAAGACATTCAGATCTTTTCTTTGATGTCCCCATTTTGTGGAGCCCATCAAAAGACCCCA CTTGTTAATCCATTAACTCGTGATGTCGTCGACAGAAGG pae_t235: ATGTATATCTCCTCCTCTCGAGTCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAG AGATACTGAGCACATCAGCAGGACGCACTGACCGAATTCATTAAAGAGGAGAAAGGTACCT AGGGATGCCTCGTGGTCGTTAACGAAAATTAACTTACTACGGGCCTTTTCTTTGATGTCCCCA TTTTGTGGAGCCCATCAAAAGGTCGAGTAGGGATGCCTCGTGGTCGTTAACGAAAATTAACT TACTACGGGACTTTTCTTTGATGTCCCCACTTTGTGGAGCCCATCAAAAGACCCCACTTGTTA ATCCATTAACTCGTGATGTTGTCGACAGAAGG 10

11 Construct name: Sequence (5 3 ) of the sense strand of the variable region shown in Fig. S1. Promoter P LTet. Promoter P LLac. Stem loop S1. Stem loop S2. Stem loop S3. his pause. TEM-1 bla orf. pae_l145: ATGTATATCTCCTCCTCTCGAGTAGGGATGCCTCGTGGTCGTTAACGAAAATTAACTTACTAC GGGACTTTTCTTTGATGTCCCCACTTTGTGGAGCCCATCAAAAGACCCCACTTGTTAATCCAT TAACTCGTGATGTTGAATTCAGTCAGTGCGTCCTGCTGATGTGCTCAGTATCTTGTTATCCGC TCACAATGTCAATTGTTATCCGCTCACAATTCTCGAGGTCGACAGAAGG pae_l174: ATGTATATCTCCTCCTCTCGAGTAGGGATGCCTCGTGGTCGTTAACGAAAATTAACTTACTAC GGGCTCTTTCAGGCGATGTGTGCTGGAAGACATTCAGATCTTTTCTTTGATGTCCCCATTTTG TGGAGCCCATCAAAAGACCCCACTTGTTAATCCATTAACTCGTGATGTCGAATTCGGTCAGT GCGTCCTGCTGATGTGCTCAGTATCTTGTTATCCGCTCACAATGTCAATTGTTATCCGCTCAC AATTCTCGAGTCGACAGAAGG pae_l235: ATGTATATCTCCTCCTCTCGAGTAGGGATGCCTCGTGGTCGTTAACGAAAATTAACTTACTAC GGGCCTTTTCTTTGATGTCCCCATTTTGTGGAGCCCATCAAAAGGTCGAGTAGGGATGCCTC GTGGTCGTTAACGAAAATTAACTTACTACGGGACTTTTCTTTGATGTCCCCACTTTGTGGAGC CCATCAAAAGACCCCACTTGTTAATCCATTAACTCGTGATGTTGAATTCAGTCAGTGCGTCCT GCTGATGTGCTCAGTATCTTGTTATCCGCTCACAATGTCAATTGTTATCCGCTCACAATTCTC GACGTCGACAGAAGG puv145placmut: ATGTATATCTCCTCCTCTCGAGTCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAG AGATACTGAGCACATCAGCAGGACGCACTGACCGAATTCATTAAAGAGGAGAAAGGTACCG GTACCTAGGGATGCCTCGTGGTCGTTAACGAAAATTAACTTACTACGGGACTTTTCTTTGATG TCCCCACTTTGTGGAGCCCATCAAAAGACCCCACTTGTTAATCCATTAACTCGTGATGTTGAA TTCAGTCAGTGCGTCCTGCTGATGTGCTCCCAGGTTTGTTATCCGCTCACAAGCCTTTTTGTT ATCCGCTCACAATTCTCGAGGTCGACAGAAGG puvbla: ATGTATATCTTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGA GGCCCTTTCGTCTTCACCTCGAGTCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATA GAGATACTGAGCACATCAGCAGGACGCACTGACCGAATTCATGAGTATTCAACATTTCCGTG TCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGT GAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTC AACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTNCCAATGATGAGCACTTT TAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTC GCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTT ACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTG CGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAAC ATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAA ACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAAC TGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAG TTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAG CCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGT ATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCG CTGAGATAGGTGCCTCACTGATTAAGCATTGGTAAGAATTCAGTCAGTGCGTCCTGCTGATG TGCTCAGTATCTTGTTATCCGCTCACAATGTCAATTGTTATCCGCTCACAATTCTCGACAGAA GG 11

12 Table S3: Parameters resulting of the Hill equation fit to the experimental data for GFP expression. * Indicates significant difference between puv145 and puv174. Only evaluated for K and H. ǂ Indicates significant difference between puv145 and puv235. Only evaluated for K and H. Indicates significant difference between puv174 and puv235. Only evaluated for K and H. 12

13 Figure S1: Plasmid construct design. (a) Plasmid map showing the original common elements in all the constructs used in this study and, in gray, the variable DNA region was modified accordingly to create the different single promoter and convergent constructs (Supporting Text 1). Sequence of the variable DNA region for each plasmid used can be found in Supporting Table S2 and depiction of the contained elements can be found in main text Fig. 2. (b) Schematic showing replacement of the original resistance marker, KanR, with AmpR, and the original origin of replication, ColE1, with p15a, using restriction enzymes AatII, XmaJI (also indicated in (a)) and SacI (see Supporting Text 2 for more details). 13

14 Figure S2: Absence of RNA interaction for complementary transcripts with an overlapping DNA length of 145 bp. (a) Schematic of sense construct pae_t145 and antisense construct pae_l145_r, producing their respective mrnas. Such system served to test for possible RNA-RNA interactions between sense and antisense complementary transcripts (b) GFP expression from pae_t145 in C600Zi at various IPTG concentrations. (c) mcherry expression from pae_l145_r in C600Zi at various IPTG concentrations. Addition of IPTG did not impact GFP expression while significantly increasing mcherry expression. Since the presence of antisense RNA does not affect expression of GFP, it can be inferred that RNA-RNA interaction was not significant in the current system. 14

15 Figure S3: Effect of P LLac activity on GFP and mcherry expression. (a) Schematic of construct puv145 with functional P LLac and mutated construct puv145placmut in which mutations at -10 and -35 P LLac regions were inserted in order to remove its transcriptional activity. (b) puv145placmut expressed 48±11% less GFP than puv145 showing the significant (p-value<0.05) positive effect of P LLac activity on GFP expression. (c) puv145placmut expressed 95±1% less mcherry than puv145 demonstrating that inserted mutations successfully removed P LLac activity. Protein expression was measured in DH5α-WT. 15

16 Figure S4: Effect of 5 UTR length on protein expression. (a) Constructs pae_t174 and pae_t235 significantly (p-value<0.05) express less GFP than pae_t145. (b) Similarly, constructs pae_l174 and pae_l235 significantly express less mcherry than pae_l145. Significant correlation between relative protein expression and 5 UTR length was not found, indicating that content of the 5 UTR has a stronger effect in protein expression than merely its length. Protein expression was measured in DH5α-WT. 16

17 References (1) Franch, T., Petersen, M., Wagner, E. G., Jacobsen, J. P., and Gerdes, K. (1999) Antisense RNA regulation in prokaryotes: rapid RNA/RNA interaction facilitated by a general U-turn loop structure. J. Mol. Biol. 294, (2) Thisted, T., Nielsen, A. K., and Gerdes, K. (1994) Mechanism of post-segregational killing: translation of Hok, SrnB and Pnd mrnas of plasmids R1, F and R483 is activated by 3 -end processing. EMBO J. 13, (3) Landick, R., Wang, D., and Chan, C. L. (1996) Quantitative analysis of transcriptional pausing by Escherichia coli RNA polymerase: his leader pause site as paradigm. Methods Enzymol. 274, (4) Lisser, S., and Margalit, H. (1993) Compilation of E. coli mrna promoter sequences. Nucleic Acids Res. 21, (5) 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 / I 1 -I 2 regulatory elements. Nucleic Acids Res. 25,