Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 104, Issue 8, Pages 2643-2648Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0606398104
Keywords
biological circuit; biopharmaceutical manufacturing; gene switch; synthetic biology; biotin
Categories
Ask authors/readers for more resources
Time-delay circuitries in which a transcription factor processes independent input parameters can modulate NF-kappa B activation, manage quorum-sensing cross-talk, and control the circadian clock. We have constructed a synthetic mammalian gene network that processes four different input signals to control either immediate or time-delayed transcription of specific target genes. BirA-mediated ligation of biotin to a biotinylation signal-containing VP16 transactivation domain triggers heterodimerization of chimeric VP16 to a streptavidin-linked tetracycline repressor (TetR). At increasing biotin concentrations up to 20 nM, TetR-specific promoters are gradually activated (off to on, input signal 1), are maximally induced at concentrations between 20 nM and 10 mu M, and are adjustably shut off at biotin levels exceeding 10 mu M (on to off, input signal 2). These specific expression characteristics with a discrete biotin concentration window emulate a biotin-triggered bandpass filter. Removal of biotin from the culture environment (input signal 3) results in time-delayed transgene expression until the intracellular biotinylated VP16 pool is degraded. Because the TetR component of the chimeric transactivator retains its tetracycline responsiveness, addition of this antibiotic (input signal 4) overrides biotin control and immediately shuts off target gene expression. Biotin-responsive immediate, bandpass filter, and time-delay transcription characteristics were predicted by a computational model and have been validated in standard cultivation settings or biopharmaceutical manufacturing scenarios using trangenic CHO-K1 cell derivatives and have been confirmed in mice. Synthetic gene circuitries provide insight into structure-function correlations of native signaling networks and foster advances in gene therapy and biopharmaceutical manufacturing.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available