Teaching an Old Bug New Tricks:

1
Teaching an Old Bug New Tricks

• Building an XOR Gate in E. coli through Promoter
  Engineering

Will DeLoache
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Synthetic Biology
Biology
Synthetic Biology
Computer Science
Biochemistry
Engineering
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Synthetic Biology
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Synthetic Biology
partsregistry.org
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Synthetic Biology
Anderson et al. Mol Sys Bio. 2007.
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Synthetic Biology
UC Berkeley iGEM, 2007
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Synthetic Biology
My Project
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BioBricks
BioBrick Part
BBa_B0015
P
S
BioBrick BioBrick plasmid backbone with some
BioBrick
origin
antibiotic resistance
BioBrick plasmid backbone
(http//partsregistry.org/Plasmids)
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BioBricks
Part A
Part B
P
S
E
origin
antibiotic resistance
origin
antibiotic resistance
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BioBricks
Part A
Part B
P
S
origin
antibiotic resistance
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BioBricks
Part A
Part B
P
S
origin
antibiotic resistance
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Part A
Part B
P
S
E
origin
antibiotic resistance
origin
antibiotic resistance
Part A
Part B
P
S
origin
antibiotic resistance
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Building an XOR Logic Gate
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Logic Gates
AND
Input A Input B Output
0 0 0
1 0 0
0 1 0
1 1 1
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Logic Gates
NAND
Input A Input B Output
0 0 1
1 0 1
0 1 1
1 1 0
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Logic Gates
OR
Input A Input B Output
0 0 0
1 0 1
0 1 1
1 1 1
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Logic Gates
XOR
Input A Input B Output
0 0 0
1 0 1
0 1 1
1 1 0
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Logic Gates
XOR
Input A Input B Output
0 0 0
1 0 1
0 1 1
1 1 0
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Quorum Sensing
http//bricker.tcnj.edu/micro/le9/Vibrio_fischeri.
jpg
Vibrio fischeri
Bobtail Squid
(Waters and Bassler, 2005)
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Quorum Sensing
3OC6
(Waters and Bassler, 2005)
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Lux and Las Systems
Lux Autoinducer
Las Autoinducer
(Waters and Bassler, 2005)
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XOR Gate Design
3OC6 3OC12 Output
0 0 0
1 0 1
0 1 1
1 1 0
LuxR3OC6
LasR3OC12
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XOR Gate Design
luxR
lasR
LuxR3OC6
LasR3OC12
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Building the XOR Promoters
pLuxLas
pLasLux
pLux
pLas
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Building the XOR Promoters
pLux
pLuxLas
134
pLux
298
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Building the XOR Promoters
Text
pLux Promoters may be unclonable
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(No Transcript)
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Fluorescence Measurements
Normalized Fluorescence Units
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Time Delayed Signal Processing
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Time Delayed Signal Processing
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Time Delayed Growth
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Time Delayed Growth
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Time Delayed Growth
4 Hours
24 Hours
48 Hours
72 Hours
6 Days
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Time Delayed Growth
Colony Growth (mm)
Time Since Plating (Hours)
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Nafcillin
Ampicillin
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37C
Colony Growth (mm)
Time Since Plating (Hours)
30C
Colony Growth (mm)
Time Since Plating (Hours)
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Conclusions

• Attempted to construct an in vivo XOR gate
• pLux promoters were unclonable
• Cross reactivity between 3OC12/LuxR
• Time Delayed Growth System
• Varied temperature, ampicillin concentration, and
  agar concentration

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Why do Synthetic Biology Research?
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Acknowledgements
Mimms Fellowship NSF (0733952) HHMI (52005120 and
52006292)James G. Martin Genomics Program
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Teaching an Old Bug New Tricks

• Building an XOR Gate in E. coli through Promoter
  Engineering

Will DeLoache
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mfold Results
pSB1A2-pLuxLas
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Bacterial Hash Function
Key 01101
Hash Value 0
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Lux Receiver
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Lab Notebook
Will DeLoache Notebook - GcatWiki
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Honors Thesis
HonorsThesis.pdf