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 APoPS Input BPoPS Output 000 100 010 111 Frameshift Suppressor tRNA PoPS Input A PoPS Input B Activator AGGA PoPS Output OFF to ON Note: This design was used by Anderson et al. (2007) with a termination suppressor tRNA

5. NAND Gate Using SSL PoPS Input APoPS Input BPoPS Output 001 101 011 110 Frameshift Suppressor tRNA PoPS Input A PoPS Input B Repressor AGGA PoPS Output ON to OFF

6. OR Gate Using SSL GACC gg CGGA Activator PoPS Input APoPS Input BPoPS Output 000 101 011 111 Frameshift Suppressor 1 Frameshift Suppressor 2 PoPS Input A PoPS Input B PoPS Output OFF to ON

7. NOR Gate Using SSL GACC gg CGGA Repressor PoPS Input APoPS Input BPoPS Output 001 100 010 110 Frameshift Suppressor 1 Frameshift Suppressor 2 PoPS Input A PoPS Input B PoPS Output OFF to ON

8. Suppressor Logic Toolkit Specific inputs and outputs

9. AND Gate Using SSL IPTG Input AaTc Input B3OC6 Output 000 100 010 111 5 bp Frameshift Suppressor tRNA plac Input A ptet Input B Activator luxO AGCGA LuxI Output OFF to ON pluxT 3OC6 aTc IPTG Other Genes Needed LacI TetR

10. NAND Gate Using SSL 3OC6 Input Apropionate Input B GFP Output 001 101 011 110 5 bp Frameshift Suppressor tRNA pluxR Input A pPrpB Input B Repressor cI AGGAC GFP Output ON to OFF pR Other Genes Needed LuxR PrpR 3OC6 Propionate

11. OR Gate Using SSL GACCT gg CGGAA Activator luxO IPTG Input AaTc Input B3OC6 Output 000 101 011 111 5 bp Frameshift Suppressor 1 5 bp Frameshift Suppressor 2 plac Input A ptet Input B 3OC6 Output OFF to ON pluxT aTc IPTG Other Genes Needed LacI TetR

12. NOR Gate Using SSL GACCT gg CGGAA Repressor cI 3OC6 Input A propionate Input B GFP Output 001 100 010 110 5 bp Frameshift Suppressor 1 5 bp Frameshift Suppressor 2 pLuxR Input A PprpB Input B GFP Output OFF to ON pR 3OC6 Propionate Other Genes Needed LuxR PrpR

13. Suppressor Logic Toolkit Experimental design

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

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

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