construction of a suppressor logic toolkit and its use in a bacterial quasi-hash function igem 2008...
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Construction of a Suppressor Logic Toolkit and its use in a Bacterial
Quasi-Hash Function
iGEM 2008 Finisher TeamBob 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– 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
Suppressor Logic Toolkit
Basic design, no specific inputs or outputs
AND Gate Using SSL
PoPS Input A PoPS Input B PoPS Output
0 0 0
1 0 0
0 1 0
1 1 1
Frameshift Suppressor tRNA
PoPS Input A
PoPS Input B
Activator
AGGAPoPS
OutputOFF to ON
Note: This design was used by Anderson et al. (2007) with a termination suppressor tRNA
NAND Gate Using SSL
PoPS Input A PoPS Input B PoPS Output
0 0 1
1 0 1
0 1 1
1 1 0
Frameshift Suppressor tRNA
PoPS Input A
PoPS Input B
Repressor
AGGAPoPS
OutputON to OFF
OR Gate Using SSL
GACC gg CGGA
Activator
PoPS Input A PoPS Input B PoPS Output
0 0 0
1 0 1
0 1 1
1 1 1
Frameshift Suppressor 1
Frameshift Suppressor 2
PoPS Input A
PoPS Input B
PoPS OutputOFF to
ON
NOR Gate Using SSL
GACC gg CGGA
Repressor
PoPS Input A PoPS Input B PoPS Output
0 0 1
1 0 0
0 1 0
1 1 0
Frameshift Suppressor 1
Frameshift Suppressor 2
PoPS Input A
PoPS Input B
PoPS OutputOFF to
ON
Suppressor Logic Toolkit
Specific inputs and outputs
AND Gate Using SSL
IPTG Input A aTc Input B 3OC6 Output
0 0 0
1 0 0
0 1 0
1 1 1
5 bp Frameshift Suppressor tRNA
plac Input A
ptet Input B Activator luxO
AGCGALuxI
OutputOFF to ON
pluxT
3OC6
aTc
IPTG
Other Genes Needed
•LacI•TetR
NAND Gate Using SSL
3OC6 Input A propionate Input B
GFP Output
0 0 1
1 0 1
0 1 1
1 1 0
5 bp Frameshift Suppressor tRNA
pluxR Input A
pPrpB Input B
Repressor cI
AGGACGFP
OutputON to OFF
pR
Other Genes Needed
•LuxR•PrpR
3OC6
Propionate
OR Gate Using SSL
GACCT gg CGGAA
Activator luxO
IPTG Input A aTc Input B 3OC6 Output
0 0 0
1 0 1
0 1 1
1 1 1
5 bp Frameshift Suppressor 1
5 bp Frameshift Suppressor 2
plac Input A
ptet Input B
3OC6 OutputOFF to
ON
pluxT
aTc
IPTG
Other Genes Needed
•LacI•TetR
NOR Gate Using SSL
GACCT gg CGGAA
Repressor cI
3OC6 Input A propionate Input B
GFP Output
0 0 1
1 0 0
0 1 0
1 1 0
5 bp Frameshift Suppressor 1
5 bp Frameshift Suppressor 2
pLuxR Input A
PprpB Input B
GFP OutputOFF to
ON
pR3OC6
Propionate
Other Genes Needed
•LuxR•PrpR
Suppressor Logic Toolkit
Experimental design
ANDcolony AmpR
NANDcolony AmpS
Biological Quasi-Hash Function
IPTG aTc
propionate
3CO6
GFP RFPor
Key message
Hash Value
Growth
ORcolony AmpR
NOR
Biological Quasi-Hash Function
IPTG aTc
propionate
3CO6
GFP RFPor
Key message
Hash Value
Growth
colony AmpS
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