Information Technology Reference
In-Depth Information
candidate genes were chosen as significantly differentially expressed. To verify
differential expression, current-induced and control RNA was electrophoresed,
blotted to nylon membranes, and hybridized with 300 basepair probes generated
from each of the candidate genes by polymerase chain reaction (PCR). Based
on Northern analysis, two genes were chosen for which
lux
reporter gene con-
structions would be made. The promoter sequences for these two genes were
amplified from the genomic DNA by PCR. We are currently cloning these
promoters into EZ:TN transposons that contain promoterless luciferase genes.
These reporter constructions will permit us to verify that the cloned pro-
moters are indeed inducible by electric current or EMF. We cannot say at this
point that these genes are specifically current inducible and not, for example,
affected by osmotic or oxidative/reductive stress. From a pragmatic point of
view, establishing specificity may not be necessary for the implementation of
current-inducible promoters in engineered devices. From this perspective it is
only necessary that the physiology of the cell is not unduly compromised during
current induction and that the linked pathways function as designed.
ELECTROCHEMICAL CONTROL OF GENE REGULATION Direct electrochemical
generation or annihilation of chemical species may be implemented for chemi-
cal regulation of gene expression. Ultimately, such electrochemical interfacing
could be implemented within individual cellular components, such that single
cells might be addressed without diffusional cross-talk to neighboring cellu-
lar components. Approaches to such interfacing may follow the direction of
electrochemical microelectrodes for cellular and subcellular chemical analyses
that have been widely reported in the literature [117] and briefly described in
a previous section of this chapter. In chapter 8 we address the construction of
nanoscale electrochemical probes that could be used for this purpose.
DESIGN, SIMULATION, AND MODELING
The design of relatively simple systems can proceed in the absence of anal-
ysis, modeling, and simulation tools. Whole-cell biosensors relying on single
reporter gene systems require only a phenomenological description of genetic
circuit function. However, more complex devices that operate through the in-
teraction of genetic circuits within an individual cell and, through cell-cell
interconnectivity, between cells, cannot be designed easily without analytical
models and simulation capabilities. The early efforts to engineer information
transport within cells described above provide the groundwork for moving
toward more complex engineered functionality of whole-cell components. Of
particular interest, the toggle switch [42] and clock circuits [35] were designed
with the aid of mathematical models, demonstrating progress toward analyti-
cal device models in the development and analysis of genetic circuits. While
