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Construction of a Suppressor Logic Toolkit and its use in a Bacterial Quasi-Hash Function

Construction of a Suppressor Logic Toolkit and its use in a Bacterial Quasi-Hash Function. iGEM 2008 Finisher Team Bob Cool, Kin Lau, Xiao Zhu. Goals for 2009 Summer Project. Build tRNA suppressor logic gate tool kit with AND, NAND, OR, and NOR logic gate functions

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Construction of a Suppressor Logic Toolkit and its use in a Bacterial Quasi-Hash Function

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  1. Construction of a Suppressor Logic Toolkit and its use in a Bacterial Quasi-Hash Function iGEM 2008 Finisher Team Bob Cool, Kin Lau, Xiao Zhu

  2. Goals for 2009 Summer Project • Build tRNA suppressor logic gate tool kit with AND, NAND, OR, and NOR logic gate functions • AND gate is same basic design as in Anderson 2007, but with different parts, including 5-base frameshift suppressor instead of termination suppressor • NAND, OR, and NOR gates are new designs • Use the logic gates to construct two distinct quasi-hash functions using cell-to-cell signaling and time-delayed growth

  3. Suppressor Logic Toolkit Basic design, no specific inputs or outputs

  4. AND Gate Using SSL PoPS Input A Frameshift Suppressor tRNA PoPS Output AGGA OFF to ON PoPS Input B Activator Note: This design was used by Anderson et al. (2007) with a termination suppressor tRNA

  5. NAND Gate Using SSL PoPS Input A Frameshift Suppressor tRNA PoPS Output AGGA ON to OFF PoPS Input B Repressor

  6. OR Gate Using SSL GACC gg CGGA PoPS Input A Frameshift Suppressor 1 Activator PoPS Output OFF to ON Frameshift Suppressor 2 PoPS Input B

  7. NOR Gate Using SSL GACC gg CGGA PoPS Input A Frameshift Suppressor ! Repressor PoPS Output OFF to ON Frameshift Suppressor 2 PoPS Input B

  8. Suppressor Logic Toolkit Specific inputs and outputs

  9. AND Gate Using SSL plac Input A pluxT IPTG 5 bp Frameshift Suppressor tRNA LuxI Output 3OC6 AGCGA OFF to ON ptet Input B aTc Activator luxO • Other Genes Needed • LacI • TetR

  10. NAND Gate Using SSL pluxR Input A 5 bp Frameshift Suppressor tRNA pcI 3OC6 GFP Output AGGAC ON to OFF pPrpB Input B Repressor cI Propionate • Other Genes Needed • LuxR • PrpR

  11. OR Gate Using SSL GACCT gg CGGAA plac Input A 5 bp Frameshift Suppressor 1 pluxT IPTG Activator luxO 3OC6 Output OFF to ON 5 bp Frameshift Suppressor 2 ptet Input B aTc • Other Genes Needed • LacI • TetR

  12. NOR Gate Using SSL GACCT gg CGGAA pLuxR Input A 5 bp Frameshift Suppressor 1 pcI 3OC6 Repressor cI GFP Output OFF to ON 5 bp Frameshift Suppressor 2 PprpB Input B Propionate • Other Genes Needed • LuxR • PrpR

  13. Suppressor Logic Toolkit Experimental design

  14. Biological Quasi-Hash Function NAND colony AmpS message Key AND colony AmpR aTc IPTG 3CO6 Growth propionate GFP RFP or Hash Value

  15. Biological Quasi-Hash Function NOR message Key OR colony AmpR aTc IPTG 3CO6 Growth propionate colony AmpS GFP RFP or Hash Value

  16. References • Anderson et al. 2007. “Environmental signal integration by a modular AND gate” http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1964800&blobtype=pdf • luxO luxT http://www.jbc.org/cgi/content/full/281/46/34775 • PprpB propionate http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16269719

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