Supplementary Information for Synthetic circuits integrating logic and memory in living cells

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1 Supplementary Information for Synthetic circuits integrating logic and memory in living cells Piro Siuti, John Yazbek and Timothy K. Lu a b AND + None AND + AHL c d AND + atc AND + atc GFP Supplementary Figure 1. Flow cytometry was used to assay the output of our recombinasebased computational units. After gating by forward and side scatter, a fluorescence threshold was applied uniformly for all logic gates to determine the percentage of cells that were deemed GFP positive (ON state) or GFP negative (OFF state). Representative flow cytometry data for the AND gate is shown here in (a) the absence of any inputs, (b) with AHL alone, (c) with atc alone, and (d) with both AHL and atc simultaneously applied. Nature Biotechnology: doi: /nbt.2510

2 a b c NOR AND XOR A B A B A B GFP GFP GFP % cells expressing GFP % cells expressing GFP % cells expressing GFP None AHL atc Both None AHL atc Both None AHL atc Both Supplementary Figure 2. The simple relationships that govern the behavior of recombinasebased circuits enable multiple instantiations of logic gates with different combinations of individual parts. In addition to the designs in Figures 1 and 2, we describe unique implementations of (a) NOR, (b) AND, and (c) XOR gates. The performance of each logic gate was characterized by the percentage of cells that were GFP positive, as assayed via flow cytometry after gating by forward and side scatter. Measurements are from three independent experiments and the error bars represent standard errors of the mean. Nature Biotechnology: doi: /nbt.2510

3 GFP Fluorescence (a.u.) proa proa_bxb1_attb proa_phic31_attb proc proc_bxb1_attb proc_phic31_attb prod prod_bxb1_attb prod_phic31_attb GFP GFP GFP Promoter Promoter_Bxb1_attB Promoter_PhiC31_attB Supplementary Figure 3. To test the effects of the variant promoters and recombinaserecognition sites on gfp expression, we characterized GFP fluoresence via flow cytometry after gating by forward and side scatter. Measurements of fluorescence values are based on geometric means from three independent experiments and the error bars represent standard errors of the mean. Nature Biotechnology: doi: /nbt.2510

4 Supplementary Figure 4. Control constructs for the digital-to-analog converters show that E. coli cells containing gfp with an inverted promoter (prod) and E. coli cells containing no gfp exhibit analog output gene expression levels comparable to the 0x outputs in Fig. 4 in the absence of both inputs (i.e., when AHL and atc inputs are both 0 ). Nature Biotechnology: doi: /nbt.2510

5 Supplementary Table 1. Synthetic parts used to implement integrated logic and memory. Part Name Part Type Sequence Promoter CACAGCTAACACCACGTCGTCCCTATCTGCTG prod CCCTAGGTCTATGAGTGGTTGCTGGATAACTT TACGGGCATGCATAAGGCTCGTATAATATATT CAGGGAGACCACAACGGTTTCCCTCTACAAAT AATTTTGTTTAACTTT proa Promoter CACAGCTAACACCACGTCGTCCCTATCTGCTG CCCTAGGTCTATGAGTGGTTGCTGGATAACTT TACGGGCATGCATAAGGCTCGTAGGCTATATT CAGGGAGACCACAACGGTTTCCCTCTACAAAT AATTTTGTTTAACTTT proc Promoter CACAGCTAACACCACGTCGTCCCTATCTGCTG CCCTAGGTCTATGAGTGGTTGCTGGATAACTT TACGGGCATGCATAAGGCTCGTATGATATATT CAGGGAGACCACAACGGTTTCCCTCTACAAAT AATTTTGTTTAACTTT T1 Unidirectional GGCATCAAATAAAACGAAAGGCTCAGTCGAA terminator AGACTGGGCCTTTCGTTTTATCTGTTGTTTGTC GGTGAACGCTCTCCTGAGTAGGACAAATCCGC CGCCCTAGA gfp Output gene ATGCGTAAAGGAGAAGAACTTTTCACTGGAGT TGTCCCAATTCTTGTTGAATTAGATGGTGATGT TAATGGGCACAAATTTTCTGTCAGTGGAGAGG GTGAAGGTGATGCAACATACGGAAAACTTACC CTTAAATTTATTTGCACTACTGGAAAACTACCT GTTCCATGGCCAACACTTGTCACTACTTTCGGT TATGGTGTTCAATGCTTTGCGAGATACCCAGA TCATATGAAACAGCATGACTTTTTCAAGAGTG CCATGCCCGAAGGTTATGTACAGGAAAGAACT ATATTTTTCAAAGATGACGGGAACTACAAGAC ACGTGCTGAAGTCAAGTTTGAAGGTGATACCC TTGTTAATAGAATCGAGTTAAAAGGTATTGAT TTTAAAGAAGATGGAAACATTCTTGGACACAA ATTGGAATACAACTATAACTCACACAATGTAT ACATCATGGCAGACAAACAAAAGAATGGAAT CAAAGTTAACTTCAAAATTAGACACAACATTG AAGATGGAAGCGTTCAACTAGCAGACCATTAT CAACAAAATACTCCAATTGGCGATGGCCCTGT CCTTTTACCAGACAACCATTACCTGTCCACAC AATCTGCCCTTTCGAAAGATCCCAACGAAAAG AGAGACCACATGGTCCTTCTTGAGTTTGTAAC AGCTGCTGGGATTACACATGGCATGGATGAAC TATACAAATAA Source Nature Biotechnology: doi: /nbt.2510

6 Bxb1 Recombinase ATGAGAGCCCTGGTAGTCATCCGCCTGTCCCG CGTCACCGATGCTACGACTTCACCGGAGCGTC AGCTGGAGTCTTGCCAGCAGCTCTGCGCCCAG CGCGGCTGGGACGTCGTCGGGGTAGCGGAGG ATCTGGACGTCTCCGGGGCGGTCGATCCGTTC GACCGGAAGCGCAGACCGAACCTGGCCCGGT GGCTAGCGTTCGAGGAGCAACCGTTCGACGTG ATCGTGGCGTACCGGGTAGACCGGTTGACCCG ATCGATCCGGCATCTGCAGCAGCTGGTCCACT GGGCCGAGGACCACAAGAAGCTGGTCGTCTCC GCGACCGAAGCGCACTTCGATACGACGACGCC GTTTGCGGCGGTCGTCATCGCGCTTATGGGAA CGGTGGCGCAGATGGAATTAGAAGCGATCAA AGAGCGGAACCGTTCGGCTGCGCATTTCAATA TCCGCGCCGGGAAATACCGAGGATCCCTGCCG CCGTGGGGATACCTGCCTACGCGCGTGGACGG GGAGTGGCGGCTGGTGCCGGACCCTGTGCAGC GAGAGCGCATCCTCGAGGTGTATCACCGCGTC GTCGACAACCACGAGCCGCTGCACCTGGTGGC CCACGACCTGAACCGGCGTGGTGTCCTGTCGC CGAAGGACTACTTCGCGCAGCTGCAAGGCCGC GAGCCGCAGGGCCGGGAGTGGTCGGCTACCG CGCTGAAGCGATCGATGATCTCCGAGGCGATG CTCGGGTACGCGACTCTGAACGGTAAGACCGT CCGAGACGACGACGGAGCCCCGCTGGTGCGG GCTGAGCCGATCCTGACCCGTGAGCAGCTGGA GGCGCTGCGCGCCGAGCTCGTGAAGACCTCCC GGGCGAAGCCCGCGGTGTCTACCCCGTCGCTG CTGCTGCGGGTGTTGTTCTGCGCGGTGTGCGG GGAGCCCGCGTACAAGTTCGCCGGGGGAGGA CGTAAGCACCCGCGCTACCGCTGCCGCTCGAT GGGGTTCCCGAAGCACTGCGGGAACGGCACG GTGGCGATGGCCGAGTGGGACGCGTTCTGCGA GGAGCAGGTGCTGGATCTGCTCGGGGACGCG GAGCGTCTGGAGAAAGTCTGGGTAGCCGGCTC GGACTCCGCGGTCGAACTCGCGGAGGTGAAC GCGGAGCTGGTGGACCTGACGTCGCTGATCGG CTCCCCGGCCTACCGGGCCGGCTCTCCGCAGC GAGAAGCACTGGATGCCCGTATTGCGGCGCTG GCCGCGCGGCAAGAGGAGCTGGAGGGTCTAG AGGCTCGCCCGTCTGGCTGGGAGTGGCGCGAG ACCGGGCAGCGGTTCGGGGACTGGTGGCGGG AGCAGGACACCGCGGCAAAGAACACCTGGCT TCGGTCGATGAACGTTCGGCTGACGTTCGACG TCCGCGGCGGGCTGACTCGCACGATCGACTTC GGGGATCTGCAGGAGTACGAGCAGCATCTCA 7 Nature Biotechnology: doi: /nbt.2510

7 Bxb1 attb Bxb1 attp Recombinase recognition site Recombinase recognition GGCTCGGCAGCGTGGTCGAACGGCTACACACC GGGATGTCGTAG TCGGCCGGCTTGTCGACGACGGCGGTCTCCGT CGTCAGGATCATCCGGGC GTCGTGGTTTGTCTGGTCAACCACCGCGGTCT CAGTGGTGTACGGTACAAACCCCGAC site phic31 Recombinase ATGACACAAGGGGTTGTGACCGGGGTGGACA CGTACGCGGGTGCTTACGACCGTCAGTCGCGC GAGCGCGAGAATTCGAGCGCAGCAAGCCCAG CGACACAGCGTAGCGCCAACGAAGACAAGGC GGCCGACCTTCAGCGCGAAGTCGAGCGCGAC GGGGGCCGGTTCAGGTTCGTCGGGCATTTCAG CGAAGCGCCGGGCACGTCGGCGTTCGGGACG GCGGAGCGCCCGGAGTTCGAACGCATCCTGAA CGAATGCCGCGCCGGGCGGCTCAACATGATCA TTGTCTATGACGTGTCGCGCTTCTCGCGCCTGA AGGTCATGGACGCGATTCCGATTGTCTCGGAA TTGCTCGCCCTGGGCGTGACGATTGTTTCCACT CAGGAAGGCGTCTTCCGGCAGGGAAACGTCAT GGACCTGATTCACCTGATTATGCGGCTCGACG CGTCGCACAAAGAATCTTCGCTGAAGTCGGCG AAGATTCTCGACACGAAGAACCTTCAGCGCGA ATTGGGCGGGTACGTCGGCGGGAAGGCGCCTT ACGGCTTCGAGCTTGTTTCGGAGACGAAGGAG ATCACGCGCAACGGCCGAATGGTCAATGTCGT CATCAACAAGCTTGCGCACTCGACCACTCCCC TTACCGGACCCTTCGAGTTCGAGCCCGACGTA ATCCGGTGGTGGTGGCGTGAGATCAAGACGCA CAAACACCTTCCCTTCAAGCCGGGCAGTCAAG CCGCCATTCACCCGGGCAGCATCACGGGGCTT TGTAAGCGCATGGACGCTGACGCCGTGCCGAC CCGGGGCGAGACGATTGGGAAGAAGACCGCT TCAAGCGCCTGGGACCCGGCAACCGTTATGCG AATCCTTCGGGACCCGCGTATTGCGGGCTTCG CCGCTGAGGTGATCTACAAGAAGAAGCCGGA CGGCACGCCGACCACGAAGATTGAGGGTTACC GCATTCAGCGCGACCCGATCACGCTCCGGCCG GTCGAGCTTGATTGCGGACCGATCATCGAGCC CGCTGAGTGGTATGAGCTTCAGGCGTGGTTGG ACGGCAGGGGGCGCGGCAAGGGGCTTTCCCG GGGGCAAGCCATTCTGTCCGCCATGGACAAGC TGTACTGCGAGTGTGGCGCCGTCATGACTTCG AAGCGCGGGGAAGAATCGATCAAGGACTCTT ACCGCTGCCGTCGCCGGAAGGTGGTCGACCCG Nature Biotechnology: doi: /nbt.2510

8 phic31 attb phic31 attp Recombinase recognition site Recombinase recognition site TCCGCACCTGGGCAGCACGAAGGCACGTGCA ACGTCAGCATGGCGGCACTCGACAAGTTCGTT GCGGAACGCATCTTCAACAAGATCAGGCACGC CGAAGGCGACGAAGAGACGTTGGCGCTTCTGT GGGAAGCCGCCCGACGCTTCGGCAAGCTCACT GAGGCGCCTGAGAAGAGCGGCGAACGGGCGA ACCTTGTTGCGGAGCGCGCCGACGCCCTGAAC GCCCTTGAAGAGCTGTACGAAGACCGCGCGGC AGGCGCGTACGACGGACCCGTTGGCAGGAAG CACTTCCGGAAGCAACAGGCAGCGCTGACGCT CCGGCAGCAAGGGGCGGAAGAGCGGCTTGCC GAACTTGAAGCCGCCGAAGCCCCGAAGCTTCC CCTTGACCAATGGTTCCCCGAAGACGCCGACG CTGACCCGACCGGCCCTAAGTCGTGGTGGGGG CGCGCGTCAGTAGACGACAAGCGCGTGTTCGT CGGGCTCTTCGTAGACAAGATCGTTGTCACGA AGTCGACTACGGGCAGGGGGCAGGGAACGCC CATCGAGAAGCGCGCTTCGATCACGTGGGCGA AGCCGCCGACCGACGACGACGAAGACGACGC CCAGGACGGCACGGAAGACGTAGCGGCGTAG TGCGGGTGCCAGGGCGTGCCCTTGGGCTCCCC GGGCGCGTACTCC GTGCCCCAACTGGGGTAACCTTTGAGTTCTCT CAGTTGGGGG 8 8 Nature Biotechnology: doi: /nbt.2510

9 Supplementary Data Nature Biotechnology: doi: /nbt.2510

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