Design and Application of Synthetic Biology Devices for Therapy - Synthetic Biology - page 162

the presence of tetracycline. G1-phase-specific growth arrest occurs when tetracycline is
withdrawn due to activation of the cycline-dependent kinase inhibitor p27 kip1 within the
synthetic gene network. Nevertheless, some cells that can escape p27 kip1 -mediated G1-phase
growth arrest spontaneously arise within the cell population at a precise frequency. This
somewhat recapitulates the natural development of cancer, whereby mitotically active cells
spontaneously appear within a growth-arrested population. Utilizing such a cellular model,
Gonzalez-Niccolini et al. 151 demonstrated that commonly used cancer drugs, such as
5-fluorouracil, doxorubicin, and etoposide, can selectively kill the spontaneously occurring
mitotic cells, while having no effect on the growth-arrested cells. Such a cellular model can
potentially be used as a first-line screening assay for potential anticancer drug candidates.

In recent years, newly emerging infectious bacterial diseases 129 and widespread antibiotic
resistance 121 have made it imperative to screen and identify new antibiotic compounds.
Aubel et al. 152 engineered a synthetic gene circuit incorporating the Streptomyces
pristinaespiralis -derived streptogramin-responsive promoter PIP into CHO cells. In response
to the streptogramin antibiotic pristinamycin I, the PIP repressor dissociates from its cognate
promoter, leading to expression of the reporter gene (secreted alkaline phosphatase).
Subsequently, the genetically engineered CHO cells were utilized for the screening and
identification of potential antibiotic candidates against clinical pathogens, which were able
to penetrate mammalian cells while exhibiting minimal cytotoxic effects.
Weber et al. 153 adopted a similar approach and developed a screening tool to identify
potential antituberculosis drug candidates that are able to penetrate mammalian cells while
having negligible cytotoxic effects. This is critical in view of the fact that Mycobacterium
tuberculosis is an intracellular pathogen. Currently, the most effective therapeutic drug against
Mycobacterium tuberculosis is ethionamide. 154 However, for ethionamide to exert its toxic
effects on Mycobacterium tuberculosis , it must be activated by Baeyer-Villiger monooxygenase
EthA within the bacterium itself. 154 Nevertheless, EthA expression is naturally repressed by
another protein, EthR. Hence, inhibitors of EthR can potentiate the therapeutic efficacy of
ethionamide on Mycobacterium tuberculosis . Therefore, the synthetic gene circuit developed by
Weber et al. 153 linked the inhibition of EthR to the expression of a reporter gene (secreted
alkaline phosphatase). Utilizing this setup, Weber et al. 153 were able to screen and identify
several cell-penetrating compounds that can inhibit EthR, while exhibiting minimal
cytotoxicity to mammalian cells.
Loose et al. 155 used linguistically based algorithms developed for syntax and grammar
analysis to examine the sequences of naturally occurring antimicrobial peptides. Certain
characteristic patterns were identified in the sequences of antimicrobial peptides, and this
information was used to design new artificial peptides that exhibit antimicrobial activity. In
this manner, the rational design of antimicrobial peptides can be achieved.

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Stem Cells and Regenerative Medicine
Synthetic biology has also found increasingly numerous applications in the field of stem
cells and regenerative medicine. Of particular interest is cellular reprogramming for the
generation of induced pluripotent stem cells (iPSC). 156 Yamanaka and colleagues first
achieved this feat in 2007 by demonstrating that differentiated somatic cells could be
reprogrammed to a pluripotent embryonic stem-cell-like state through recombinant
expression of four genes KLF4, OCT4, c-MYC, and SOX2. 157 This groundbreaking study

has made it possible to derive immunocompatible cells of any lineage for transplantation/
transfusion therapy.

Nevertheless, the major challenge faced in cellular reprogramming for the generation of iPSC
is the use of recombinant DNA, with its attendant risk of permanent genetic modification to
the cellular genome. One strategy to overcome this challenge would be to transiently insert