Supplementary Information

Transcription

1 Supplementary Information RapGene: a fast and accurate strategy for synthetic gene assembly in Escherichia coli Massimiliano Zampini, Pauline Rees Stevens, Justin A. Pachebat, Alison Kingston-Smith, Luis A. J. Mur & Finbarr Hayes

2 Supplementary Figure S1. prg1.0 assembly and structure (not to scale). Plasmid prg1.0 was assembled from two PCR products derived from pgata using the Gibson Assembly Cloning Kit. psm, streptomycin/spectinomycin promoter from pcdfduet-1. ptac, tac promoter. RBS, ribosome binding site. Olac, lac operator. bla, beta-lactamase gene. ori, origin of replication. laci q, lac repressor gene with a modified promoter. NheI, restriction site for NheI., restriction site for. End-overlaps, homology regions required by the Gibson assembly cloning kit.

3 psm NheI GAATAAAAAAGAAAAAGGAGAgctagc CTTATTTTTTCTTTTTCCTCTcgatcg GST-Gata1 ptac cttaaggttgttggtgggtgca gaattccaacaaccacccacgt + (NheI + + T4 DNA polymerase + dttps) GAATAAAAAAGAAAAAGGAGAg CTTATTTTTTCTTTTTCCTCTcgatc ttaaggttgttggtgggtgca ccaacaaccacccacgt GAAT CTTATTTTTTCTTTTTCCTCTcgatc ttaaggttgttggtgggtgca T GAAT AAAAAAGAAAAAGGAGAgctagcATGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTAA ttaaggttgttggtgggtgca CTTATTTTTTCTTTTTCCTCTcgatc TACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNATTgaattcCAACAACCACCCACG T Synthetic gene Supplementary Figure S2. prg1.0 cloning region (not to scale). Plasmid prg1.0 is prepared to generate long 3 -recessed cohesive ends by digesting with and NheI in the presence of T4 DNA polymerase and 1 mm dttps. The reaction is stopped by spin-column purification. The single-nucleotide gap (boxed on the complementary strand) between the assembled synthetic insert and the vector, produced in all inserts after annealing, is filled in vivo in E. coli. Dashed lines represent the single oligonucleotides within the synthetic gene.

4 Name Sequence (5-3 ) PCR1f PCR1r PCR2f PCR2r Sm-1 Sm-2 Sm-3 Sm-4 Sm-5 Sm-6 Sm-7 Sm-8 Sm-9 Sm-10 Sm-11 ACAGCTCActtaagGTTGTTGGTGGGTGCAGCCGGAAGCATAAAGTGT AAAGCC CCTAGGTTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAAT GTATTTAGAAAAATACCGGTACCTCTGACACATGCAGCTCCCGGAG TCATGAGACAATAACCCTGATAAACCTAGGAATAAAAAAGAAAAAGG AGAgctagcTTTCACCGTCATCACCGAAACGCGCGAGGCAGATCGTCA G TGCACCCACCAACAACcttaagTGAGCTGTTGACAATTAATCATCGGCT CG AAAAAAGAAAAAGGAGAgctagcATGAGGGAAGCGGTGATCGCCGAAG TATCGACTCAAC GACGCCAACTACCTCTGATAGTTGAGTCGATACTTCGGCGATCACCG CTTCCCTCAT TATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTT GCTGGCCGTACA CCACTGCGGAGCCGTACAAATGTACGGCCAGCAACGTCGGTTCGAG ATGGCGCTCGAT TTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGAT ATTGATTTGCTG AGCCTTACGGTCACCGTAACCAGCAAATCAATATCACTGTGTGGCTT CAGGCCGCCAT GTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGAT CAACGACCTTT AGGGGAAGCCGAAGTTTCCAAAAGGTCGTTGATCAAAGCTCGCCGC GTTGTTTCATCA TGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGT AGAAGTCACCAT TGATGTCGTCGTGCACAACAATGGTGACTTCTACAGCGCGGAGAATC TCGCTCTCTCC TGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCG AACTGCAATTT Restriction sites NheI NheI Signals - promoter (-35 and -10 regions) - promoter (-10 region) Notes Template for PCR: pgata Template for PCR: pgata Template for PCR: pgata Template for PCR: pgata

5 Sm-12 TCATTGCGCTGCCATTCTCCAAATTGCAGTTCGCGCTTAGCTGGATA ACGCCACGGAA Sm-13 GGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAG CCACGATCGACA Sm-14 CAGCAAGATAGCCAGATCAATGTCGATCGTGGCTGGCTCGAAGATAC CTGCAAGAATG Sm-15 TTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCC TTGGTAGGTCC Sm-16 CAAAGAGTTCCTCCGCCGCTGGACCTACCAAGGCAACGCTATGTTCT CTTGCTTTTGT Sm-17 AGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTG AGGCGCTAAAT Sm-18 TTCCATAGCGTTAAGGTTTCATTTAGCGCCTCAAATAGATCCTGTTCA GGAACCGGAT Sm-19 GAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATG AGCGAAATGTAG Sm-20 AATGCGGGACAACGTAAGCACTACATTTCGCTCATCGCCAGCCCAGT CGGGCGGCGAG Sm-21 TGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATC GCGCCGAAGGA Sm-22 TTGCCCAGTCGGCAGCGACATCCTTCGGCGCGATTTTGCCGGTTACT GCGCTGTACCA Sm-23 TGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAG CCCGTCATACTT Sm-24 AGATAAGCCTGTCTAGCTTCAAGTATGACGGGCTGATACTGGGCCG GCAGGCGCTCCA Sm-25 GAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTC GCGCGCAGATC Sm-26 GTGGACAAATTCTTCCAACTGATCTGCGCGCGAGGCCAAGCGATCTT CTTCTTGTCCA Sm-27 AGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTA GTCGGCAAATAA - aada1 stop codon Sm-28 GCACCCACCAACAACcttaagTTATTTGCCGACTACCTTGGTGATCTCG CCTTTCACGTA - aada1 stop codon Sm-A1 GCACCCACCAACAACcttaagCAGCAAATCAATATCACTGTGTGGCTTC

6 Sm-A2 Sm-A3 Sm-A AGGCCGCCAT GCACCCACCAACAACcttaagAAATTGCAGTTCGCGCTTAGCTGGATAA CGCCACGGAA GCACCCACCAACAACcttaagATTTAGCGCCTCAAATAGATCCTGTTCA GGAACCGGAT GCACCCACCAACAACcttaagAAGTATGACGGGCTGATACTGGGCCGG CAGGCGCTCCA AAAAAAGAAAAAGGAGAgctagcATGAGTAAAGGAGAAGAACTTTTCAC TGGAGTTGTCCCAATTC TTTGCCCATTAACATCGCCATCTAATTCAACAAGAATTGGGACAACTC CAGTGAAAAGTTCTTCTCCTTTACTCAT TTGTTGAATTAGATGGCGATGTTAATGGGCAAAAATTCTCTGTCAGTG GAGAGGGTGAAGGTGATG AAATAAATTTAAGGGTAAGTTTTCCGTATGTTGCATCACCTTCACCCT CTCCACTGACAGAGAATT CAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGGAAGC TACCTGTTCCATGGCCAA ATTGAACACCATAAGAGAAAGTAGTGACAAGTGTTGGCCATGGAACA GGTAGCTTCCCAGTAGTGC CACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACC CAGATCATATGAAACAGC AACCTTCGGGCATGGCACTCTTGAAAAAGTCATGCTGTTTCATATGAT CTGGGTATCTTGAAAAGC ATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGA ACTATATTTTACAAAGATG ACTTGACTTCAGCACGTGTCTTGTAGTTCCCGTCATCTTTGTAAAATA TAGTTCTTTCCTGTACAT ACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACC CTTGTTAATAGAATCGAGT TGTTTCCATCTTCTTTAAAATCAATACCTTTTAACTCGATTCTATTAAC AAGGGTATCACCTTCAA TAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAAA TGGAATACAACTATAACT TTGGTTTGTCTGCCATGATGTATACATTATGTGAGTTATAGTTGTATTC CATTTTGTGTCCAAGAA NheI

7 RG1.0f RG1.0 r CACATAATGTATACATCATGGCAGACAAACCAAAGAATGGAATCAAA GTTAACTTCAAAATTAGAC CTGCTAATTGAACGCTTCCATCTTTAATGTTGTGTCTAATTTTGAAGTT AACTTTGATTCCATTCT ACAACATTAAAGATGGAAGCGTTCAATTAGCAGACCATTATCAACAAA ATACTCCAATTGGCGATG ACAGGTAATGGTTGTCTGGTAAAAGGACAGGGCCATCGCCAATTGGA GTATTTTGTTGATAATGGT GCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCC CTTTCCAAAGATCCCAACG CAAACTCAAGAAGGATCATGTGATCTCTCTTTTCGTTGGGATCTTTGG AAAGGGCAGATTGTGTGG AAAAGAGAGATCACATGATCCTTCTTGAGTTTGTAACAGCTGCTGGG ATTACACATGGCATGGATGAACTATACAAATAA GCACCCACCAACAACcttaagTTATTTGTATAGTTCATCCATGCCATGTG TAATCCCAGCAGCTGTTA CTCCGGGAGCTGCATG GGCACGACAGGTTTCCCG gfp stop codon gfp stop codon Supplementary Table S1. Oligonucleotides used in this study. Sequences designed to anneal to the DNA template in PCR are underlined. Boxed regions correspond to the homology sequences required by the Gibson Assembly Cloning Kit to produce prg1.0. Sequences highlighted in black represents signals (i.e. promoters, stop codons). Restriction sites are indicated in lower case.

8 aada1-assembly Type of assembly E. coli strain sample Cycle 0 Cycle 1 Cycle 2 aada1 (6-oligonucleotides) 100% 100% 90% aada1 (12-oligonucleotides) 100% 100% 100% aada1 (18-oligonucleotides) 80% 70% 70% aada1 (24-oligonucleotides) - 20% - aada1 (28-oligonucleotides) - 20% 10% gfp-assembly Type of assembly E. coli strain sample Cycle 0 Cycle 1 Cycle 2 Oligonucleotides pre-annealed separately in three groups gfp (22-oligonucleotides) 44% gfp (22-oligonucleotides) - 20% 15% 15% 20% 15% 10% Supplementary Table S2. RapGene aada1 and gfp cloning efficiencies during the first two freezing-thawing cycles of the oligonucleotides used for the corresponding assemblies. Percentages of correct-size inserts as determined by colony PCR are reported. The table also reports yields for the assembly performed with three groups of consecutive oligonucleotides pre-annealed separately. - -