Engineering E. coli strains using antibiotic-resistance-gene-free plasmids.

We created a generalizable pipeline for antibiotic-resistance-gene-free plasmid (ARGFP)-based cloning using a dual auxotrophic- and essential-gene-based selection strategy. We use auxotrophic selection to construct plasmids in engineered E. coli DH10B cloning strains and both auxotrophic- and essential-gene-based selection to (1) select for recombinant strains and (2) maintain a plasmid in E. coli Nissle 1917, a common chassis for engineered probiotic applications, and E. coli MG1655, the laboratory "wild-type" E. coli strain. We show that our approach has comparable efficiency to that of antibiotic-resistance-gene-based cloning. We also show that the double-knockout Nissle and MG1655 strains are simple to transform with plasmids of interest. Notably, we show that the engineered Nissle strains are amenable to long-term plasmid maintenance in repeated culturing as well as in the mouse gut, demonstrating the potential for broad applications while minimizing the risk of antibiotic resistance spread via horizontal gene transfer.

Characterizing a Propionate Sensor in E. coli Nissle 1917.

Short-chain fatty acids (SCFAs) are commonly found in the large intestine, but generally not in the small intestine, and influence microbiome composition and host physiology. Thus, synthetic biologists are interested in developing engineered probiotics capable of in situ detection of SCFAs as biogeography or disease sensors. One SCFA, propionate, is both sensed and consumed by E. coli. Here, we utilize the E. coli transcription factor PrpR, sensitive to the propionate-derived metabolite (2S,3S)-2-methylcitrate, and its cognate promoter PprpBCDE to detect extracellular propionate with the probiotic chassis bacterium E. coli Nissle 1917. We identify that PrpR-PprpBCDE displays stationary phase leakiness and transient bimodality, and we explain these observations through evolutionary rationales and deterministic modeling, respectively. Our results will help researchers build biogeographically sensitive genetic circuits.

Engineering ligand-specific biosensors for aromatic amino acids and neurochemicals.

Microbial biosensors have diverse applications in metabolic engineering and medicine. Specific and accurate quantification of chemical concentrations allows for adaptive regulation of enzymatic pathways and temporally precise expression of diagnostic reporters. Although biosensors should differentiate structurally similar ligands with distinct biological functions, such specific sensors are rarely found in nature and challenging to create. Using E. coli Nissle 1917, a generally regarded as safe microbe, we characterized two biosensor systems that promiscuously recognize aromatic amino acids or neurochemicals. To improve the sensors' selectivity and sensitivity, we applied rational protein engineering by identifying and mutagenizing amino acid residues and successfully demonstrated the ligand-specific biosensors for phenylalanine, tyrosine, phenylethylamine, and tyramine. Additionally, our approach revealed insights into the uncharacterized structure of the FeaR regulator, including critical residues in ligand binding. These results lay the groundwork for developing kinetically adaptive microbes for diverse applications. A record of this paper's transparent peer review process is included in the supplemental information.

Engineering microbial diagnostics and therapeutics with smart control.

Microbes have become an increasingly powerful chassis for developing diagnostic and therapeutic technologies. While many of the earlier engineering efforts used microbes that expressed relevant proteins constitutively, more microbes are being engineered to express them with region-selectivity and disease-responsiveness through biosensors. Such 'smart' microbes have been developed to diagnose and treat a wide range of disorders and diseases, including bacterial infections, cancers, inflammatory disorders, and metabolic disorders. In this review, we discuss synthetic biology technologies that have been applied to engineer microbes for biomedical applications, focusing on recent reports that demonstrate microbial sensing by using animal models or clinical samples. Advances in synthetic biology will enable engineered microbes to significantly improve the medical field.

Biosensing in Smart Engineered Probiotics.

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.

Modulating Responses of Toehold Switches by an Inhibitory Hairpin.

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.

Establishing a Multivariate Model for Predictable Antisense RNA-Mediated Repression.

Recent advances in our understanding of RNA folding and functions have facilitated the use of regulatory RNAs such as synthetic antisense RNAs (asRNAs) to modulate gene expression. However, despite the simple and universal complementarity rule, predictable asRNA-mediated repression is still challenging due to the intrinsic complexity of native asRNA-mediated gene regulation. To address this issue, we present a multivariate model, based on the change in free energy of complex formation (Δ GCF) and percent mismatch of the target binding region, which can predict synthetic asRNA-mediated repression efficiency in diverse contexts. First, 69 asRNAs that bind to multiple target mRNAs were designed and tested to create the predictive model. Second, we showed that the same model is effective predicting repression of target genes in both plasmids and chromosomes. Third, using our model, we designed asRNAs that simultaneously modulated expression of a toxin and its antitoxin to demonstrate tunable control of cell growth. Fourth, we tested and validated the same model in two different biotechnologically important organisms: Escherichia coli Nissle 1917 and Bacillus subtilis 168. Last, multiple parameters, including target locations, the presence of an Hfq binding site, GC contents, and gene expression levels, were revisited to define the conditions under which the multivariate model should be used for accurate prediction. Together, 434 different strain-asRNA combinations were tested, validating the predictive model in a variety of contexts, including multiple target genes and organisms. The result presented in this study is an important step toward achieving predictable tunability of asRNA-mediated repression.

Robust production of recombinant phosphoproteins using cell-free protein synthesis.

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 signalling 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 signalling networks and kinase inhibitor drugs.