Biology Reference
In-Depth Information
and for application in regenerative medicine. However, tinkering with the biological
functions of bacteria and viruses can also have useful clinical applications. The diverse
population of commensal and symbiotic microorganisms associated with the human body,
known as the human microbiome, forms a complex ecosystem that plays an important role
in regulating human physiology, health, and disease states.
26,27
The fact that the human body
naturally tolerates the various species of microorganisms associated with it makes these
microorganisms ideal targets for synthetic biology applications. Indeed, synthetic gene
circuits have been engineered in human microbiome-associated bacterial species to confer
host resistance to infectious diseases such as cholera,
28
as well as to kill cancerous cells.
29
Besides bacterial cells, synthetic gene circuits can also be deployed in viruses for therapeutic
applications. Of particular interest are the bacteriophages, which are viruses that specifically
infect bacterial cells. The T7 phage has been engineered to express the bacterial biofilm-
degrading enzyme dispersin B,
30
while a synthetic gene circuit that interferes with the SOS
response network in bacterial cells has been engineered in the M13 phage.
31
MOLECULAR TOOLKIT FOR SYNTHETIC BIOLOGY
Synthetic Gene Circuits Encoded by Recombinant DNA
GENE TARGETING AND GENOME EDITING TECHNOLOGIES
Transfection of recombinant DNA into mammalian or bacterial cells is an essential
prerequisite for the deployment of synthetic gene circuits. The major challenge is to achieve
efficient and site-specific integration of the transfected recombinant DNA into the host
genome. Transient transfection with nonintegrating plasmid DNA imposes severe
limitations on long-term clinical applications. Moreover, there is a low probability of
random and nonsite-specific integration of the plasmid DNA into the host genome, which
could cause insertional mutagenesis of host genes. This could potentially lead to cancer, and
hence invoke serious safety concerns.
32
Although the use of viral vectors can efficiently
integrate recombinant DNA into the host genome, again, the problem is the random and
nonsite-specific integration of recombinant DNA into the cellular host genome.
33,34
160
The development of site-specific recombinase (SSR) technology was the first attempt at site-
specific manipulation of genomic DNA. SSR systems such as Cre-LoxP,
35
Flp-FRT,
36
Dre,
37
and PhiC31
38
can be used to delete, insert, or invert a segment of DNA flanked by specific
recombination sites within genomic DNA, and have now been incorporated into viral
vectors.
39,40
More recently, there have been advances in the rational design of zinc finger
(ZF) nucleases for site-specific insertion of recombinant DNA.
41
44
The nonspecific Fok1
domain of ZF nucleases can be coupled to transcription activator-like effectors (TALEs) to
form novel genome-editing tools that enable precise integration of recombinant DNA into
target chromosomal locations.
45
It would also be of clinical interest to be able to transiently insert and remove transgenic
elements in a precise and site-specific manner, without leaving any permanent modification
to genomic DNA. For example, some synthetic biology applications may require temporary
expression of synthetic gene circuits within cells for only a limited period of time. The
PiggyBac transposon system can be particularly useful for this purpose.
46,47
Wilson et al.
48
demonstrated that PiggyBac integration and excision within human genomic DNA is very
precise, without leaving any
mutations at the site of transposon excision. The
same study
48
also mapped a total of 575 PiggyBac integration sites within human genomic
DNA to demonstrate the nonrandom site-selectivity of PiggyBac transposon integration.
Such useful properties of the PiggyBac transposon system have been utilized for
reprogramming mammalian somatic cells to pluripotent stem cells through the transient
expression of four transgenes (c-Myc, Klf4, Oct4, and Sox2), without leaving any permanent
genetic alteration to the reprogrammed cells.
49,50
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