Understanding the functional and structural consequences of site-specific protein phosphorylation has remained limited by our inability to produce phosphoproteins at high yields. Here, we address this limitation by developing a cell-free protein synthesis (CFPS) platform that employs crude extracts from a genomically recoded strain of Escherichia coli for site-specific, co-translational incorporation of phosphoserine into proteins. We apply this system to the robust production of up to milligram quantities of human MEK1 kinase. Then, we recapitulate a physiological signaling cascade in vitro to evaluate the contributions of site-specific phosphorylation of mono- and doubly-phosphorylated forms on MEK1 activity. We discover that only one phosphorylation event is necessary and sufficient for MEK1 activity. Our work sets the stage for using CFPS as a rapid high-throughput technology platform for direct expression of programmable phosphoproteins containing multiple phosphorylated residues. This work will facilitate study of phosphorylation-dependent structure-function relationships, kinase signaling networks, and kinase inhibitor drugs.
Engineered microbes are exciting alternatives to current diagnostics and therapeutics. Researchers have developed a wide range of genetic tools and parts to engineer probiotic and commensal microbes. Among these tools and parts, biosensors allow the microbes to sense and record or to sense and respond to chemical and environmental signals in the body, enabling them to report on health conditions of the animal host and/or deliver therapeutics in a controlled manner. This review focuses on how biosensing is applied to engineer "smart" microbes for in vivo diagnostic, therapeutic, and biocontainment goals. Hurdles that need to be overcome when transitioning from high-throughput in vitro systems to low-throughput in vivo animal models, new technologies that can be implemented to alleviate this experimental gap, and areas where future advancements can be made to maximize the utility of biosensing for medical applications are also discussed. As technologies for engineering microbes continue to be developed, these engineered organisms will be used to address many medical challenges.
The
toehold switch consists of a cis-repressing switch
RNA hairpin and a trans-acting trigger RNA. The binding
of the trigger RNA to an unpaired toehold sequence of the switch hairpin
allows for a branch migration process, exposing the start codon and
ribosome binding site for translation initiation. In this work, we
demonstrate that responses of toehold switches can be modulated by
introducing an inhibitory hairpin that shortens the length of the
unpaired toehold region. First, we investigated the effect of the
toehold region length on output gene expression and showed that the
second trigger RNA, which binds to the inhibitory hairpin, is necessary
for output gene activation when the hairpin-to-hairpin spacing is
short. Second, the apparent Hill coefficient was found generally to
increase with decreasing hairpin-to-hairpin spacing or increasing
hairpin number. This work expands the utility of toehold switches
by providing a new way to modulate their response.
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