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Revision as of 15:11, 17 October 2018
Primers used
Primers
gBlocks
> alphaCD19(CO) tgaaccgtcagatccgctagcgctaccggtGCCACCATGGCGCTGCCGGTCACGGCATTG CTCCTTCCGTTGGCCCTGCTCTTGCATGCCGCTAGACCCTACCCCTATGATGTACCAGAT TACGCGGATATACAAATGACCCAGACTACGTCCAGCCTTTCCGCCTCCCTGGGTGATCGC GTGACAATCAGTTGTAGGGCTAGTCAGGACATAAGTAAATACCTCAACTGGTATCAACAG AAGCCTGACGGAACAGTTAAACTTCTTATCTATCATACAAGTAGGCTTCATTCAGGCGTT CCATCCAGATTTAGTGGATCTGGGAGCGGCACCGACTACTCACTCACGATTAGTAATCTG GAACAAGAGGACATCGCCACTTATTTTTGCCAGCAGGGTAACACTCTTCCCTATACATTC GGCGGCGGTACGAAACTCGAAATTACAGGGGGGGGTGGCAGTGGTGGCGGAGGTTCCGGT GGGGGTGGTTCAGAAGTAAAACTTCAGGAATCTGGTCCAGGGCTGGTTGCACCCAGCCAA TCCCTTTCCGTCACGTGCACCGTATCCGGGGTCTCCCTCCCAGACTACGGCGTGAGTTGG ATACGACAGCCGCCGCGCAAGGGGCTTGAGTGGCTGGGTGTCATTTGGGGATCTGAAACA ACGTATTATAATTCCGCTTTGAAATCCCGCCTCACTATTATCAAGGACAATTCTAAGAGC CAGGTCTTTCTGAAGATGAACAGCCTGCAAACGGATGACACAGCCATCTACTATTGTGCA AAGCACTATTACTATGGTGGTTCATACGCTATGGATTACTGGGGACAAGGGACATCCGTA ACCGTTTCATCACCCTGCGTCGGTAGCAACCC
> tTAA(CO) TGTCACGAAAGCGCCGACGGATGAGTCGATTGGATAAATCTAAGGTAATTAACAGCGCAC TGGAGCTTCTGAACGAAGTAGGTATCGAGGGGCTCACCACACGAAAATTGGCACAGAAGC TCGGAGTGGAGCAGCCGACTTTGTACTGGCACGTTAAGAATAAGAGGGCTTTGCTTGACG CGCTGGCCATTGAGATGCTCGACCGGCATCACACTCACTTCTGCCCGCTTGAAGGCGAGA GTTGGCAAGATTTCCTTAGGAACAATGCAAAGAGCTTTCGATGCGCCCTGCTTAGCCATC GGGACGGCGCAAAGGTTCATCTTGGGACTCGGCCTACTGAGAAGCAATATGAAACCCTGG AAAACCAGCTTGCTTTCCTTTGCCAACAGGGGTTCTCACTGGAGAATGCCCTGTACGCAC TCTCCGCGGTCGGCCATTTTACTCTCGGGTGTGTTTTGGAAGATCAGGAACACCAGGTAG CGAAAGAGGAGCGAGAAACGCCTACAACTGATTCCATGCCACCGTTGCTGAGACAAGCTA TAGAGTTGTTCGACCACCAGGGTGCTGAACCGGCGTTTCTCTTTGGTCTGGAACTCATTA TCTGCGGCCTTGAAAAGCAGCTTAAGTGCGAGAGTGGCGGTCCCGCAGATGCACTTGATG ACTTTGATTTGGATATGTTGCCCGCGGACGCTCTTGATGACTTCGATCTGGATATGCTGC CGGCTGATGCCCTCGACGATCTTGACCTGGATATGCTCCCAGGATGAaagcttggaaccc ttaatataacttcgtat
> TetOn3G(CO) GGAGGTgaattcGCCACCATGAGCCGACTGGACAAGAGCAAGGTAATCAATTCAGCTTTG GAGCTTCTGAACGGCGTGGGAATCGAGGGTCTCACCACCAGAAAGCTCGCGCAAAAATTG GGGGTGGAACAACCGACTCTCTATTGGCATGTTAAGAACAAGAGGGCCCTCCTTGACGCA CTCCCCATCGAAATGCTGGATCGACATCACACACATAGTTGTCCGCTGGAGGGGGAGTCT TGGCAAGATTTCCTCAGGAATAACGCGAAGAGTTATCGGTGCGCTCTCCTTAGTCATCGC GATGGAGCGAAAGTGCATCTGGGGACAAGACCGACGGAAAAACAATATGAGACGTTGGAG AACCAACTGGCATTCTTGTGCCAACAGGGGTTCAGCCTTGAAAATGCTCTTTACGCCTTG AGCGCTGTTGGGCACTTCACCTTGGGCTGTGTACTGGAGGAGCAGGAGCACCAGGTCGCA AAGGAGGAGCGCGAAACGCCCACCACAGATAGTATGCCACCCCTGTTGAAGCAGGCTATC GAGCTTTTTGATCGGCAGGGTGCTGAGCCTGCCTTTTTGTTCGGCCTTGAGCTTATAATT TGCGGCCTGGAAAAACAACTCAAGTGCGAAAGCGGTGGTCCCACAGACGCATTGGACGAT TTTGATCTTGACATGCTGCCAGCCGATGCTTTGGACGATTTTGACCTGGATATGTTGCCG GCAGATGCCCTCGACGACTTTGATCTTGATATGTTGCCGGGCTAAggatccGGAGGT
> mN1ce(CO) CCCTGCGTCGGTAGCAACCCCTGCTACAATCAAGGGACCTGCGAGCCAACGTCAGAAAAT CCCTTTTATAGATGCCTGTGCCCGGCAAAGTTCAACGGACTGCTGTGCCACATACTCGAC TACAGTTTTACTGGGGGGGCGGGCCGGGATATACCCCCGCCTCAGATAGAAGAGGCATGC GAACTGCCGGAATGCCAGGTCGATGCGGGCAATAAAGTCTGCAACCTCCAATGCAATAAT CACGCCTGCGGTTGGGACGGGGGTGACTGCTCACTTAACTTCAATGATCCGTGGAAGAAC TGCACCCAGTCCCTCCAATGTTGGAAATATTTTTCCGATGGTCATTGTGATAGCCAGTGC AATTCCGCAGGATGTTTGTTCGATGGGTTTGACTGCCAACTGACAGAGGGTCAGTGTAAT CCTCTTTACGACCAATACTGCAAGGATCATTTCAGTGATGGACATTGTGACCAAGGTTGT AACAGTGCGGAGTGCGAATGGGATGGGTTGGATTGTGCTGAGCATGTTCCTGAGCGCCTC GCAGCGGGAACTTTGGTCCTCGTTGTCCTCCTTCCGCCTGACCAGCTCAGAAATAATAGC TTTCACTTTCTTAGAGAACTCTCTCATGTGTTGCATACCAACGTAGTCTTTAAGCGCGAC GCTCAAGGACAGCAAATGATCTTCCCGTACTATGGGCACGAAGAAGAGCTTCGGAAACAC CCGATAAAAAGGAGTACGGTTGGTTGGGCGACGAGTAGTCTTTTGCCGGGCACGAGTGGT GGCAGACAACGGCGGGAACTCGATCCGATGGACATACGGGGGTCCATAGTGTATCTGGAG ATTGACAACCGACAGTGTGTTCAAAGTTCAAGCCAGTGTTTTCAATCAGCCACTGATGTA GCTGCGTTTCTCGGAGCGTTGGCCTCTCTTGGGAGCTTGAATATTCCGTATAAAATCGAA GCTGTAAAAAGCGAACCTGTCGAACCGCCATTGCCAAGCCAGTTGCACCTCATGTACGTG GCCGCTGCTGCGTTCGTACTCTTGTTCTTCGTAGGCTGCGGTGTTTTGCTGTCACGAAAG CGCCGACGG
> hN1c(CO) CCCTGTCTTGGAGGGAATCCGTGTTACAACCAAGGGACATGTGAACCCACGAGCGAGTCT CCTTTTTACAGGTGTCTCTGTCCAGCAAAATTTAATGGATTGCTTTGCCACATTCTGGAT TACTCATTTGGTGGGGGAGCGGGGCGGGACATACCGCCGCCCCTTATTGAAGAAGCATGC GAACTGCCCGAGTGCCAGGAGGATGCAGGTAACAAGGTATGCTCTCTTCAGTGCAATAAC CACGCTTGCGGGTGGGATGGTGGCGATTGCTCCTTGAATTTTAATGATCCTTGGAAAAAT TGCACCCAATCACTTCAGTGTTGGAAATACTTCTCAGATGGCCATTGCGATTCTCAGTGT AACAGCGCGGGATGCTTGTTTGACGGATTCGACTGTCAAAGGGCTGAGGGACAATGTAAT CCGCTCTACGATCAGTATTGTAAAGACCATTTCAGCGACGGCCACTGTGACCAAGGCTGC AATAGTGCCGAGTGTGAGTGGGATGGGTTGGATTGCGCGGAGCATGTTCCGGAGAGACTT GCGGCAGGTACGCTCGTGGTTGTTGTACTCATGCCGCCTGAGCAACTGAGGAATAGTAGC TTTCACTTCCTCCGGGAACTGAGCCGGGTTCTGCATACTAACGTTGTATTTAAGAGAGAT GCACATGGTCAGCAAATGATATTTCCGTACTACGGTCGGGAAGAGGAATTGCGGAAGCAC CCCATTAAGAGAGCTGCCGAGGGTTGGGCCGCTCCGGATGCCCTCCTTGGACAAGTAAAA GCCTCCCTCCTTCCCGGCGGATCTGAGGGTGGACGGAGACGCCGGGAACTCGATCCAATG GATGTAAGAGGGTCCATTGTGTACTTGGAGATTGATAATCGCCAGTGTGTGCAGGCGTCC TCACAATGTTTTCAAAGTGCGACCGATGTCGCAGCATTCTTGGGTGCCCTGGCATCATTG GGCAGCCTGAATATTCCATATAAGATCGAAGCGGTGCAATCCGAAACGGTGGAACCGCCT CCACCGGCACAGCTTCATTTCATGTATGTCGCAGCAGCAGCTTTCGTTTTGCTTTTCTTC GTAGGGTGTGGTGTTCTCCTTAGTCGAAAACGACGCCGG
> LaG17(CO) tgaaccgtcagatccgctagcgctaccggtGCCACCATGGCGCTGCCGGTCACGGCATTG CTCCTTCCGTTGGCCCTGCTCTTGCATGCCGCTAGACCCGAGCAAAAGTTGATCTCAGAG GAGGACCTCATGGCCGATGTGCAGCTCGTCGAGTCAGGTGGGGGTCTCGTACAAGCTGGG GGGTCACTCAGACTGTCTTGCGCCGCCAGTGGCAGAACCATCTCTATGGCAGCTATGTCC TGGTTCAGACAGGCGCCAGGCAAGGAGAGGGAGTTTGTGGCGGGTATAAGTAGGTCCGCA GGAAGCGCGGTCCACGCCGATTCAGTCAAAGGTCGGTTCACGATAAGCCGGGATAACACT AAGAACACTCTTTATCTCCAGATGAACTCTCTGAAGGCCGAGGATACGGCTGTTTACTAT TGCGCTGTACGGACATCTGGGTTCTTCGGGAGCATACCGCGCACCGGAACAGCGTTTGAT TACTGGGGACAAGGTACACAGGTGACCGTTTCCCCCTGCGTCGGTAGCAACCC
> LaG16-2(CO) tgaaccgtcagatccgctagcgctaccggtGCCACCATGGCGCTGCCGGTCACGGCATTG CTCCTTCCGTTGGCCCTGCTCTTGCATGCCGCTAGACCCGAGCAAAAGTTGATCTCAGAG GAGGACCTCATGGCACAGGTCCAACTCGTCGAATCCGGAGGTCGCCTCGTGCAAGCTGGC GATTCACTTCGACTCTCTTGCGCGGCGTCAGGTCGGACCTTCAGCACGTCAGCTATGGCG TGGTTCCGCCAGGCGCCTGGGAGAGAGAGAGAGTTTGTAGCAGCCATCACATGGACTGTT GGAAATACTATATTGGGCGACTCCGTAAAAGGGCGCTTCACTATCTCACGAGATAGAGCG AAAAACACTGTAGATCTGCAAATGGACAATTTGGAGCCGGAGGATACTGCTGTGTATTAT TGCAGCGCTCGCTCTCGGGGGTACGTCCTCTCCGTACTCCGGAGCGTGGATTCATACGAC TACTGGGGACAGGGCACACAAGTAACGGTAAGCGGTGGGGGAGGGTCTGGTGGTGGAGGC TCAGGAGGCGGGGGATCTATGGCTCAGGTCCAGCTTGTAGAGAGCGGTGGTGGATTGGTG CAAGCAGGAGGGAGCCTCAGGCTTAGTTGTGCTGCCTCCGGTCGAACCTTCTCAAATTAT GCAATGGGTTGGTTTAGGCAAGCTCCTGGTAAAGAACGGGAGTTCGTGGCCGCAATTAGC TGGACGGGAGTTAGCACATATTACGCTGACTCTGTGAAAGGTCGGTTCACGATAAGTCGA GACAATGATAAGAATACGGTTTACGTCCAAATGAATAGCCTCATCCCGGAGGACACAGCT ATATATTATTGCGCTGCTGTACGGGCAAGATCCTTCTCAGATACTTATAGCAGGGTTAAC GAATACGATTATTGGGGGCAGGGAACGCAAGTCACTGTTCCCTGCGTCGGTAGCAACCC
> EGFPtm(CO) GGAAGAAAATCCCGGTCCGATGGCCCTTCCTGTAACGGCGCTCCTTCTCCCTCTTGCCC TCCTGCTCCATGCCGCACGACCAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGG TGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCG AGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCA AGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCA GCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCT ACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGG TGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGG AGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA TCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCG AGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCC CCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCA ACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCG GCATGGACGAGCTGTACAAGAATGCCGTTGGTCAAGACACGCAGGAAGTCATTGTTGTAC CCCACTCCCTGCCATTTAAGGTGGTAGTAATTTCAGCCATCCTTGCTCTCGTTGTGTTGA CAATTATTTCTCTGATAATTCTTATCATGCTCTGGCAAAAGAAACCTCGCTGAaagcttg gaacccttaatataacttcgtat
> CD19ECD(CO) TTGCTCCCACTGGCCTTGCTGCTTCATGCGGCACGGCCTTACCCCTATGATGTACCAGAT TACGCGGAAGAGCCTCTCGTCGTGAAAGTTGAAGAAGGTGACAATGCAGTTTTGCAATGT CTGAAAGGGACGTCTGACGGACCAACCCAACAGCTCACTTGGAGTCGCGAATCCCCGCTT AAGCCGTTCCTGAAGTTGTCTCTGGGGTTGCCGGGTCTTGGTATACACATGCGCCCGCTT GCGATATGGCTCTTCATCTTCAATGTCAGCCAACAGATGGGCGGGTTCTATCTCTGTCAA CCGGGACCGCCATCAGAGAAGGCGTGGCAACCTGGTTGGACGGTAAACGTGGAAGGTAGC GGGGAGTTGTTTCGCTGGAACGTTTCTGATCTCGGGGGCCTGGGTTGCGGTCTGAAAAAT CGCTCTAGCGAAGGGCCGAGTTCACCTTCCGGCAAGCTCATGTCCCCGAAACTCTACGTG TGGGCGAAGGATAGGCCAGAAATTTGGGAGGGAGAACCCCCCTGCCTGCCGCCCAGAGAT TCACTCAACCAGAGTCTCTCCCAAGATCTGACTATGGCCCCAGGAAGTACCTTGTGGCTT AGCTGCGGTGTACCGCCCGACTCCGTGAGCCGAGGGCCTCTCTCTTGGACACACGTTCAC CCTAAGGGGCCGAAATCATTGTTGAGTTTGGAACTCAAAGACGATAGGCCAGCAAGGGAT ATGTGGGTTATGGAGACCGGCCTTCTGCTCCCACGAGCGACCGCTCAGGATGCGGGCAAG TATTATTGCCACAGAGGAAATCTGACTATGTCTTTTCATCTGGAAATTACTGCGCGGCCA GTTTTGTGGCACTGGCTGTTGCGCACGGGGGGATGGAAGGAGCAGAAATTGATCTCTGA
> Gal4DBD(CO) atgaagctgctgagcagcatcgagcaggcctgtgacatctgccggctgaagaaactgaag tgcagcaaagaaaagcccaagtgcgccaagtgcctgaagaacaactgggagtgccggtac agccccaagaccaagagaagccccctgaccagagcccacctgaccgaggtggaaagccgg ctggaaagactggaacagctgtttctgctgatcttcccacgcgaggacctggacatgatc ctgaagatggacagcctccaggacatcaaggccctgctgaccggcctgttcgtgcaggac aacgtgaacaaggacgccgtgaccgacagactggccagcgtggaaaccgacatgcccctg accctgcggcagcacagaatcagcgccaccagcagcagcgaggaaagcagcaacaagggc cagcggcagctgacagtgtct
> ZF21.16(CO) GCTAGCCCTAGAGTGAGAACAGGTTCTAAGACACCTCCTCACGAGAGGCCTTTTCAATGT AGAATTTGTATGCGTAATTTTTCTaggaacttcatcctccagagaCATACAAGGACTCAC ACAGGTGAAAAGCCTTTTCAGTGCAGAATTTGTATGCGCAATTTTTCTcagggcggtaac ctggtgagaCATCTGCGTACCCACACTGGTGAAAAGCCTTTCCAATGTAGAATTTGTATG CGAAATTTTTCTcagcaaaccggcctgaacgtgCATCTGAAGACACACACAGGCGAGAAG GGATCC
> ZF42.10(CO) GCTAGCCCTAGAGTGAGAACAGGTTCTAAGACACCACCACATGAAAGACCATTCCAATGT AGAATTTGTATGAGGAATTTTTCTaccggccagatcctggacagaCATACAAGGACCCAC ACCGGCGAAAAGCCATTCCAGTGTAGAATTTGTATGCGTAATTTTTCTgtggcccacagc ctgaagagaCATCTGAGGACCCACACCGGAGAGAAGCCTTTTCAATGCAGAATTTGTATG CGTAATTTTTCTgaccccagcaacctgagaaggCATCTGAAGACCCACACCGGAGAAAAG GGATCC
> ZF43.8(CO) GCTAGCCCCAGAGTGAGAACCGGTTCTAAGACACCTCCCCACGAGAGGCCTTTTCAGTGT AGAATTTGTATGCGTAATTTTTCTaggcaggacagactggacaggCACACAAGGACTCAC ACTGGTGAAAAGCCCTTCCAATGTAGAATTTGTATGCGCAATTTTTCTcagaaggagcac ctggccggcCATCTGAGGACCCACACAGGCGAAAAGCCTTTCCAGTGCAGAATTTGTATG AGAAATTTTTCTaggagagacaacctgaacagaCATCTGAAGACACATACAGGTGAAAAA GGATCC
> TEVp(FL)(CO) ATCACGTTAGGGCTAGTCGACGCCACCATGGGGGAGAGCCTGTTCAAAGGGCCCCGCGAT TATAACCCTATAAGCAGTACAATATGTCATCTGACTAACGAATCAGACGGACATACGACC TCCCTCTACGGGATAGGGTTCGGCCCCTTCATCATCACCAATAAGCACTTGTTTAGGCGA AATAACGGCACGCTTCTGGTACAGTCCCTGCACGGGGTCTTTAAGGTCAAGAATACGACA ACTCTCCAACAGCATCTTATTGATGGACGCGACATGATTATCATTAGAATGCCTAAAGAT TTTCCGCCGTTCCCACAAAAATTGAAGTTTAGAGAACCGCAAAGGGAAGAGCGCATTTGT TTGGTAACCACTAATTTTCAGACAAAGAGCATGTCCTCCATGGTTAGTGACACTTCATGC ACGTTTCCTTCTTCTGATGGTATATTTTGGAAACATTGGATACAGACGAAAGACGGACAG TGCGGGTCCCCTCTTGTCTCTACCAGGGACGGCTTTATCGTCGGCATACATTCAGCCAGT AATTTCACAAACACAAATAATTATTTTACTAGCGTGCCCAAAAATTTCATGGAGTTGCTG ACCAATCAAGAGGCACAGCAGTGGGTTTCTGGATGGAGACTCAACGCCGACAGCGTACTC TGGGGTGGACATAAGGTGTTCATGgtgAAACCCGAGGAACCCTTTCAGCCGGTGAAAGAG GCCACGCAACTGATGAACGAATTGGTCTATAGCCAGTGAAAGCTTCCCTCATTCAAGTAC
> ZF21_16N-CfaN-CfaC-ZF21_16C(CO) ATCACGTTAGGGCTAGTCGACGCCACCatgGCAAGTCCTAGGGTGCGGACCGGATCAAAG ACGCCGCCTCACGAACGACCATTTCAATGCAGGATCTGCATGCGGAACTTCTCCCGGAAC TTTATCCTCCAGCGGCATACACGGACCCATACCGGAGAAAAGCCCTTCCAGTGCCTCTCA TATGACACGGAAATTTTGACCGTCGAATATGGCTTTCTTCCTATCGGGAAGATCGTTGAA GAAAGAATTGAATGCACGGTCTATACCGTGGATAAGAATGGTTTTGTATACACTCAACCG ATAGCGCAGTGGCATAACAGGGGGGAGCAAGAAGTCTTTGAATACTGTTTGGAGGATGGG TCCATCATCAGGGCAACCAAGGACCATAAGTTCATGACCACTGATGGGCAGATGCTTCCG ATTGATGAAATTTTCGAAAGGGGCCTTGACCTGAAGCAGGTCGATGGGTTGCCAATGGTT AAGATCATATCACGGAAGAGCCTGGGGACACAGAATGTGTATGATATTGGAGTAGGCGAG CCGCACAACTTCCTCCTTAAAAATGGTCTCGTAGCTTCAAACTGTAGGATATGCATGCGA AACTTCTCCCAGGGGGGCAACTTGGTCAGGCACCTTCGGACGCATACCGGAGAGAAGCCA TTTCAGTGCAGGATTTGCATGCGAAATTTTTCTCAGCAAACCGGGTTGAATGTACATCTG AAAACGCATACAGGTGAGAAGGGCTCCTGAAAGCTTCCCTCATTCAAGTAC
Abstract
Contact-dependent signaling is critical for multicellular biological events, yet customizing contact-dependent signal transduction between cells remains challenging. Here we have developed the ENABLE toolbox, a complete set of transmembrane binary logic gates. Each gate consists of 3 layers: Receptor, Amplifier, and Combiner. We first optimized synthetic Notch receptors to enable cells to respond to different signals across the membrane reliably. These signals, individually amplified intracellularly by transcription, are further combined for computing. Our engineered zinc finger-based transcription factors perform binary computation and output designed products. In summary, we have combined spatially different signals in mammalian cells, and revealed new potentials for biological oscillators, tissue engineering, cancer treatments, bio-computing, etc. ENABLE is a toolbox for constructing contact-dependent signaling networks in mammals. The 3-layer design principle underlying ENABLE empowers any future development of transmembrane logic circuits, thus contributes a foundational advance to Synthetic Biology.