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.

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