Biocomputing: in vivo AND gate

1
Biocomputing in vivo AND gate J.R. Willard,
M.L. Simpson, B. Applegate, J. Flemming, G. Sayler
Abstract Living cells have an amazing capacity
for storing information on their genome. This
memory has the potential for use in information
storage and processing. The construction of an
in vivo AND logic gate is a step toward
information processing by genetically-engineered
whole cells. In our model system, luminescence
will be induced by the presence of both
tetracycline and homoserine lactone. The
presence of tetracycline will cause the removal
of a repressor from the operator (Otet) that
controls both the tetracycline resistance gene
(tetR) and the lux regulator gene (luxR).
Homoserine lactone will then bind to the LuxR
protein, which will result in the expression of
the luxCDABE genes and ultimately in the
production of light. Either inducer on its own
should result in no light production. The AND
gate is under construction in plasmids in
Escherichia coli, and will be moved into
Pseudomonas fluorescens where the system will be
tested for luminescence.

• Future Research
•  
• Transposition of genes into P. fluorescens
• Generation of a truth table using P. fluorescens
  mutants showing that luminescence only occurs in
  the presence of both tetracycline and homoserine
  lactone
• Linking of in vivo AND gate with in vivo XOR gate
  (E. Senning) to demonstrate computational
  properties of the system by using multiple inputs
  to obtain a final output

Results
TN5TcNX/T2/tetR/Otet
pUTK215

• Introduction
• Developing in vivo logic gates is one way to
  demonstrate that we can tap into the information
  processing capabilities of whole cells
• Using the tools of molecular biology, an AND gate
  can be constructed in P. fluorescens
• Functionality of the AND gate exhibits
  information processing in bacteria

Operation of final construct

• Methods
• Isolate genomic DNA from Vibrio fischeri
• Isolate by PCR luxR
• TA clone luxR, digest with Not I/Spe I, insert
  into Not I/Xba I cut TA vector containing
  Otet/tetR
• Isolate by PCR Otet/tetR/luxR genes, adding a Not
  I site, TA clone, digest with Not I/Xba I, insert
  into TN5TcNX/T2/tetR/Otet
• Insert tetR/Otet/luxR genes by transposition into
  P. fluorescens
• Isolate by PCR Olux from V. fischeri genomic DNA
• TA clone lux operator, digest with Not I/Spe I,
  insert into Not I/Xba I cut pUTK215 vector
• Insert genes by transposition into P. fluorescens

• Objectives
• Generate plasmids in E. coli with relevant genes
  luxR, Otet, tetR, luxCDABE, and Olux
• Move genes by transposition into P. fluorescens
• Demonstrate that P. fluorescens are dependent on
  the presence of tetracycline and homoserine
  lactone for luminescence

Acknowledgements   The Department of Energy ERULF
program provided the opportunity to conduct this
research. Thank you to Dr. Michael Simpson,
Principal Investigator, for advice and placement
on this project. Thank you to the University of
Tennessee Center for Environmental Biotechnology
(UT-CEB), where all of this research was
conducted, and to Professor Gary Sayler, Director
of the UT-CEB. Thanks to Dr. Bruce Applegate for
unending patience in guidance. Finally thank you
to Eric Senning.