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1.
J Biol Chem ; 300(5): 107248, 2024 May.
Article in English | MEDLINE | ID: mdl-38556082

ABSTRACT

P2X receptors are a family of ligand gated ion channels found in a range of eukaryotic species including humans but are not naturally present in the yeast Saccharomyces cerevisiae. We demonstrate the first recombinant expression and functional gating of the P2X2 receptor in baker's yeast. We leverage the yeast host for facile genetic screens of mutant P2X2 by performing site saturation mutagenesis at residues of interest, including SNPs implicated in deafness and at residues involved in native binding. Deep mutational analysis and rounds of genetic engineering yield mutant P2X2 F303Y A304W, which has altered ligand selectivity toward the ATP analog AMP-PNP. The F303Y A304W variant shows over 100-fold increased intracellular calcium amplitudes with AMP-PNP compared to the WT receptor and has a much lower desensitization rate. Since AMP-PNP does not naturally activate P2X receptors, the F303Y A304W P2X2 may be a starting point for downstream applications in chemogenetic cellular control. Interestingly, the A304W mutation selectively destabilizes the desensitized state, which may provide a mechanistic basis for receptor opening with suboptimal agonists. The yeast system represents an inexpensive, scalable platform for ion channel characterization and engineering by circumventing the more expensive and time-consuming methodologies involving mammalian hosts.


Subject(s)
Receptors, Purinergic P2X2 , Saccharomyces cerevisiae , Humans , Amino Acid Substitution , Ligands , Protein Engineering/methods , Receptors, Purinergic P2X2/metabolism , Receptors, Purinergic P2X2/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/genetics , Models, Molecular , Protein Structure, Tertiary , Protein Structure, Quaternary , Structural Homology, Protein , Mutation
2.
ACS Synth Biol ; 12(12): 3771-3777, 2023 Dec 15.
Article in English | MEDLINE | ID: mdl-38050859

ABSTRACT

The incorporation of unnatural amino acids is an attractive method for improving or bringing new and novel functions in peptides and proteins. Cell-free protein synthesis using the Protein Synthesis Using Recombinant Elements (PURE) system is an attractive platform for efficient unnatural amino acid incorporation. In this work, we further adapted and modified the One Pot PURE to obtain a robust and modular system for enzymatic single-site-specific incorporation of an unnatural amino acid. We demonstrated the flexibility of this system through the introduction of two different orthogonal aminoacyl tRNA synthetase:tRNA pairs that suppressed two distinctive stop codons in separate reaction mixtures.


Subject(s)
Amino Acids , Amino Acyl-tRNA Synthetases , Amino Acids/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolism , Proteins/genetics , Amino Acyl-tRNA Synthetases/metabolism , Codon, Terminator/genetics
3.
Plant Methods ; 18(1): 42, 2022 Mar 29.
Article in English | MEDLINE | ID: mdl-35351174

ABSTRACT

BACKGROUND: The construction and application of synthetic genetic circuits is frequently improved if gene expression can be orthogonally controlled, relative to the host. In plants, orthogonality can be achieved via the use of CRISPR-based transcription factors that are programmed to act on natural or synthetic promoters. The construction of complex gene circuits can require multiple, orthogonal regulatory interactions, and this in turn requires that the full programmability of CRISPR elements be adapted to non-natural and non-standard promoters that have few constraints on their design. Therefore, we have developed synthetic promoter elements in which regions upstream of the minimal 35S CaMV promoter are designed from scratch to interact via programmed gRNAs with dCas9 fusions that allow activation of gene expression. RESULTS: A panel of three, mutually orthogonal promoters that can be acted on by artificial gRNAs bound by CRISPR regulators were designed. Guide RNA expression targeting these promoters was in turn controlled by either Pol III (U6) or ethylene-inducible Pol II promoters, implementing for the first time a fully artificial Orthogonal Control System (OCS). Following demonstration of the complete orthogonality of the designs, the OCS was tied to cellular metabolism by putting gRNA expression under the control of an endogenous plant signaling molecule, ethylene. The ability to form complex circuitry was demonstrated via the ethylene-driven, ratiometric expression of fluorescent proteins in single plants. CONCLUSIONS: The design of synthetic promoters is highly generalizable to large tracts of sequence space, allowing Orthogonal Control Systems of increasing complexity to potentially be generated at will. The ability to tie in several different basal features of plant molecular biology (Pol II and Pol III promoters, ethylene regulation) to the OCS demonstrates multiple opportunities for engineering at the system level. Moreover, given the fungibility of the core 35S CaMV promoter elements, the derived synthetic promoters can potentially be utilized across a variety of plant species.

4.
PLoS One ; 16(6): e0252507, 2021.
Article in English | MEDLINE | ID: mdl-34061896

ABSTRACT

We recently developed 'cellular' reagents-lyophilized bacteria overexpressing proteins of interest-that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.


Subject(s)
COVID-19 Testing/standards , COVID-19/diagnosis , COVID-19/epidemiology , Diagnostic Tests, Routine/standards , Indicators and Reagents/standards , Real-Time Polymerase Chain Reaction/standards , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Testing/methods , Cameroon/epidemiology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Geobacillus stearothermophilus/genetics , Geobacillus stearothermophilus/metabolism , Ghana/epidemiology , Humans , Indicators and Reagents/chemistry , Indicators and Reagents/metabolism , Indicators and Reagents/supply & distribution , Molecular Diagnostic Techniques , Plasmids/chemistry , Plasmids/metabolism , Real-Time Polymerase Chain Reaction/methods , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Synthetic Biology/methods , Transformation, Bacterial , United Kingdom/epidemiology
5.
ACS Synth Biol ; 7(8): 1937-1945, 2018 08 17.
Article in English | MEDLINE | ID: mdl-30021064

ABSTRACT

In vitro transcription networks are analogs of naturally occurring gene regulatory networks that consist of synthetic DNA templates that are cross-regulated by their own transcripts. This ability to design and execute in vitro transcription networks has allowed bottom-up construction of complex network topologies with predictable dynamic behavior. Here we describe the simplified design of an in vitro transcription network based on single-stranded synthetic DNA hairpin switches that function similar to molecular beacons, via toehold mediated strand displacement. Systematic construction of increasingly larger circuits was achieved by programming interactions between multiple switches through rational sequence design, and the dynamic behavior of networks was accurately predicted using a simple mathematical model. Ultimately, we engineered a cascade of switches that acted as a Boolean complete NAND gate capable of sensing both DNA and RNA inputs. The tools and framework that have been developed makes the execution of in vitro transcription circuits much simpler, which will enable them to more readily serve as testbeds for nucleic acid computations both in vitro and in vivo.


Subject(s)
Gene Regulatory Networks/genetics , DNA/genetics , Escherichia coli/genetics , Nucleic Acids/genetics , Promoter Regions, Genetic/genetics , Transcription, Genetic/genetics
6.
ACS Synth Biol ; 7(5): 1279-1290, 2018 05 18.
Article in English | MEDLINE | ID: mdl-29608282

ABSTRACT

Engineering the bacteria present in animal microbiomes promises to lead to breakthroughs in medicine and agriculture, but progress is hampered by a dearth of tools for genetically modifying the diverse species that comprise these communities. Here we present a toolkit of genetic parts for the modular construction of broad-host-range plasmids built around the RSF1010 replicon. Golden Gate assembly of parts in this toolkit can be used to rapidly test various antibiotic resistance markers, promoters, fluorescent reporters, and other coding sequences in newly isolated bacteria. We demonstrate the utility of this toolkit in multiple species of Proteobacteria that are native to the gut microbiomes of honey bees ( Apis mellifera) and bumble bees (B ombus sp.). Expressing fluorescent proteins in Snodgrassella alvi, Gilliamella apicola, Bartonella apis, and Serratia strains enables us to visualize how these bacteria colonize the bee gut. We also demonstrate CRISPRi repression in B. apis and use Cas9-facilitated knockout of an S. alvi adhesion gene to show that it is important for colonization of the gut. Beyond characterizing how the gut microbiome influences the health of these prominent pollinators, this bee microbiome toolkit (BTK) will be useful for engineering bacteria found in other natural microbial communities.


Subject(s)
Bees/microbiology , Gastrointestinal Microbiome/genetics , Genetic Engineering/methods , Proteobacteria/genetics , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Expression Regulation, Bacterial , Ileum/microbiology , Microorganisms, Genetically-Modified , Plasmids , Promoter Regions, Genetic , Replicon , Serratia marcescens/genetics , Serratia marcescens/pathogenicity , Symbiosis
7.
Methods ; 143: 110-120, 2018 07 01.
Article in English | MEDLINE | ID: mdl-29518499

ABSTRACT

T7 RNA polymerase (T7 RNAP) is one of the preferred workhorses for recombinant gene expression, owing in part to its high transcriptional activity and the fact that it has a small (17 base-pair), easily manipulated promoter. Furthermore, the fact that T7 RNAP is largely orthogonal to most hosts enables its use in a wide variety of contexts. However, the high activity of the enzyme also often leads to an increased fitness burden on the host, limiting the predictability of its interactions and impact on host physiology, and potentially leading to mutations in the constructs. Here we use a synthetic biology approach to design and characterize a panel of T7 RNAP expression circuits with different modes of regulation that enable the reliable expression of downstream targets under a variety of conditions. First, we describe the construction of a minimal T7 RNAP expression system that is inducible by a small molecule anhydrotetracycline (aTc), and then characterize a self-limiting T7 RNAP expression circuit that provides better control over the amount of T7 RNAP produced upon induction. Finally, we characterize a so-called T7 RNAP homeostasis circuit that leads to constitutive, continuous, and sub-toxic levels of T7 RNAP. Coupled with previously characterized mutant T7 RNAP promoters in vitro, we demonstrate that this modular framework can be used to achieve precise and predictable levels of output (sfGFP) in vivo. This new framework should now allow modeling and construction of T7 RNAP expression constructs that expand the utility of this enzyme for driving a variety of synthetic circuits and constructs.


Subject(s)
Cloning, Molecular/methods , DNA-Directed RNA Polymerases/metabolism , Promoter Regions, Genetic/genetics , Transcription, Genetic , Viral Proteins/metabolism , DNA-Directed RNA Polymerases/genetics , Electroporation/instrumentation , Electroporation/methods , Escherichia coli , Green Fluorescent Proteins/genetics , Models, Biological , Mutation , Plasmids/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Synthetic Biology/instrumentation , Synthetic Biology/methods , Transformation, Bacterial , Viral Proteins/genetics
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