The Development of an XOR Logic Gate in Pseudomonas fluorescens E' N' Senning, M' L' Simpson, B' App

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The Development of an XOR Logic Gate in
Pseudomonas fluorescensE. N. Senning, M. L.
Simpson, B. Applegateand G. S. Sayler
Abstract Along with Jennifer Willard and
researchers at ORNL and UTs Center for
Environmental Biotechnology (CEB), I will explore
the possibility of creating logic gates within
Pseudomonas fluorescens cells. More specifically,
I have been assigned the project of creating an
exclusive OR (XOR) logic gate. An XOR gate has
two input ports and one output port. If either of
the two input ports is logic true, the XOR output
will be logic true. However, if both inputs are
logic true or both are logic false, the XOR
output is logic false. Case 1 Case
2 Input(0,0)------gtOutput(0)
Input(0,1)------gtOutput(1) Case
3 Case 4 Input(1,0)------gtOutput(1)
Input(1,1)------gtOutput(0) Gene inserts and
gene regulation will provide the backbone to the
logic gate. Regulating the expression of an
inserted luxAB gene in Pseudomonas fluorescens
cells with the use of promoter molecules will be
the means of computing with each cell. The luxAB
gene encodes an enzyme that then breaks down an
aldehyde in a process producing light.

• Objectives
• This projects objective is to design an in vivo
  XOR gate embedded in the genome of Pseudomonas
  fluorescens.
• The illustration depicted below shows the
  mechanism by which light is emitted as an output
  from an XOR logic gate. Luciferase is the enzyme
  that oxidizes myristal aldehyde in a light
  emitting reaction.
• In the model our lab has developed, salicylate
  is simulated by compound A. Salicylate induces
  expression of the nahR gene near the promoter
  region, which induces transcription of the upper
  sense-antisense mRNA.
• Diagram 2. is analogues to the upper regulatory
  pathway (1.). However, Compound B simulates
  toluene, and the activated regulatory genes are
  todS and T, which also induce transcription of
  the second mRNA.
• As are the expectations of an XOR logic gate, the
  presence of both salicylate and toluene do not
  allow an engineered cell to produce light. This
  is portrayed in scheme 3.

• Accomplishments to Date
• The salicylate activated gene insert has been
  completed and is being prepared for transposition
  into the genome of Pseudomonas fluorescens.
• To the right, E.coli carrying the salicylate
  (compound A) gene insert in a plasmid are assayed
  for luminescence after exposure to different
  chemical stimulants.
• Difficulties have been confronted in the
  synthesis of the second sense-antisense insert,
  which is to be activated by toluene. The addition
  of the sense and antisense extensions to either
  side of the luxAB has been problematic.

• Summary
• The nahR gene insert (activated by salicylate)
  shows a high level of latent activity as
  indicated in the graph above. However, the high
  copy number of the plasmid carrying this insert
  inside E. coli provides a totally different
  situation compared to when the insert is actually
  within the genome of Pseudomonas fluorescens.
• Should the sense-antisense XOR gate fail to
  work, a second type of XOR gate is also feasible
  with repressor proteins controlling the gene
  expression of luxAB.

Introduction Logic gates are already
pre-existing structures in the world of
electronics, and the advent of biomolecular
engineering has opened up the possibility of
pursuing logic systems in biological cells. Cells
are very sensitive to the constitution of their
environment. For example, the presence of a toxic
molecule, even in minute concentrations, can be
very influential on the cells ability to
function properly. By means of manipulating the
genetic information of a healthy cell,
researchers at the UT Center for Environmental
Bioengineering have made it possible to detect
the presence of chemical compounds through
engineered cells. In the process of detecting the
compound, the cells then express a protein that
can catalyze the degradation of myristaldehyde to
produce light. The bioluminescence of cells has a
direct correlation to the concentration of the
target chemical. Observing the bioluminescence
of such cells as described in the previous
paragraph can serve as an indicator to the
presence of the compound to which the cell is
made sensitive. This causal relationship between
the affecting molecule (chemical compound) and
the reporting event (light) still applies to the
development of logic gates. The XOR logic gate
can be activated by either of two stimuli and
must exhibit one type of response. Furthermore,
the response of one cell becomes the affecting
molecule in another engineered cell, and since
each logic gate cell has more than one input, a
series of logic gates can be implemented to
actually compute.
Acknowledgements The ORSS program for
undergraduate students The Department of
Energy The Center of Environmental Biotechnology
at UT Knoxville