Expression System Research Articles

Binding properties of chemosensory protein 4 in Riptortus pedestris to aggregation pheromones

In insects, chemosensory proteins (CSPs) play an important role in the perception of the external environment and have been widely used for protein-binding characterization. Riptortus pedestris has received increased attention as a potential cause of soybean staygreen syndrome in recent years. In this study, we found that RpedCSP4 expression in the antennae of adult R. pedestris increased with age, with no significant difference in expression level observed between males and females, as determined through quantitative real-time polymerase chain reaction (qRT-PCR). Subsequently, we investigated the ability of RpedCSP4 to bind various ligands (five aggregated pheromone components and 13 soybean volatiles) using a prokaryotic expression system and fluorescence competitive binding assays. We found that RpedCSP4 binds to three aggregated pheromone components of R. pedestris, namely, ((E)-2-hexenyl (Z)-3-hexenoate (E2Z3), (E)-2-hexenyl (E)-2-hexenoate (E2E2), and (E)-2-hexenyl hexenoate (E2HH)), and that its binding capacities are most stable under acidic condition. Finally, the structure and protein-ligand interactions of RpedCSP4 were further analyzed via homology modeling, molecular docking, and targeted mutagenesis experiments. The L29A mutant exhibited a loss of binding ability to these three aggregated pheromone components. Our results show that the olfactory function of RpedCSP4 provides new insights into the binding mechanism of RpedCSPs to aggregation pheromones and contributes to discover new target candidates that will provide a theoretical basis for future population control of R. pedestris.

Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing

BackgroundThe abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers.ResultsThe aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-β-xylanase and β-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA β-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L− 1 after 48 h under oxygen limited condition in bioreactor fermentations.ConclusionThis work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast’s expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.

Construction of prokaryotic system for expression of porcine circovirus type 2 ORF-2 gene fragment

Porcine circovirus-associated diseases (PCVDs) are among the most significant challenges for pig farming in developed countries. Porcine circovirus type 2 (PCV-2) is considered the main etiological agent of postweaning multisystemic wasting syndrome in piglets. Mass PCVD occurrence has been reported in most regions of the world, that results in serious economic consequences. Optimal PCVD prevention is known to be achieved through a set of veterinary and sanitary measures in combination with vaccination. High evolutionary virus variability facilitating new genotype and strain emergence requires development of new candidate recombinant vaccines against PCV-2 infection. The study was aimed at construction of prokaryotic system for PCV-2 ORF-2 gene fragment expression and its functionality assessment. A genetic insert constructed from the most immunogenic type-specific PCV-2 epitopes based on genotype 2a, 2b, 2d strain and isolate consensus sequence was cloned into the expression vector pET-22b(+) that was incorporated into the Escherichia coli strain Rosetta 2(DE3). The transformants were selected based on the marker gene of ampicillin resistance on a selective medium. Target gene expression was induced by adding of isopropyl-β-D-1-thiogalactopyranoside at different concentrations. As a result, Escherichia coli Rosetta 2(DE3)/pET-22b-ORF-2 strain, a producer of capsid protein fragment (92–233 amino acid residues), was constructed. It was found that in the presence of 1 mM isopropyl-β-D-1-thiogalactopyranoside, the expression level of soluble truncated rCap was 35–40 mg/L 6 hours after induction. The expression product was tested for its specificity with indirect ELISA using whole-virion PCV-2-hyperimmunized porcine serum. It was shown that the positivity coefficient of producer strain cell lysates averaged to 4.34 (p < 0.005). The recombinant rCap protein is suitable for serological diagnosis and is also of interest as a vaccine component, which is the goal of our further studies.

Filamentous fungus-produced human monoclonal antibody provides protection against SARS-CoV-2 in hamster and non-human primate models

Monoclonal antibodies are an increasingly important tool for prophylaxis and treatment of acute virus infections like SARS-CoV-2 infection. However, their use is often restricted due to the time required for development, variable yields and high production costs, as well as the need for adaptation to newly emerging virus variants. Here we use the genetically modified filamentous fungus expression system Thermothelomyces heterothallica (C1), which has a naturally high biosynthesis capacity for secretory enzymes and other proteins, to produce a human monoclonal IgG1 antibody (HuMab 87G7) that neutralises the SARS-CoV-2 variants of concern (VOCs) Alpha, Beta, Gamma, Delta, and Omicron. Both the mammalian cell and C1 produced HuMab 87G7 broadly neutralise SARS-CoV-2 VOCs in vitro and also provide protection against VOC Omicron in hamsters. The C1 produced HuMab 87G7 is also able to protect against the Delta VOC in non-human primates. In summary, these findings show that the C1 expression system is a promising technology platform for the development of HuMabs in preventive and therapeutic medicine.

Identification and functional characterization of chemosensory genes in olfactory and taste organs of Spodoptera litura (Lepidoptera: Noctuidae).

The tobacco cutworm Spodoptera litura is one of the most destructive polyphagous crop pests. Olfaction and taste play a crucial role in its host plant selection and sexual communication, but the expression profile of chemosensory genes remains unclear. In this study, we identified 185 chemosensory genes from 7 organs in S. litura by transcriptome sequencing, of which 72 genes were published for the first time, including 27 odorant receptors (ORs), 26 gustatory receptors (GRs), 1 ionotropic receptor (IR), 16 odorant-binding proteins (OBPs), and 2 chemosensory proteins (CSPs). Phylogenetic analyses revealed that ORs, IRs, OBPs, and sensory neuron membrane proteins (SNMPs) were mainly expressed in antennae and sequence-conserved among Noctuidae species. The most differentially expressed genes (DEGs) between sexes were ORs and OBPs, and no DEGs were found in GRs. GR transcripts were enriched in proboscis, and the expression of sugar receptors was the highest. Carbon dioxide receptors, sugar receptor-SliuGR6, and bitter GRs-SlituGR43 and SlituGR66 had higher sequence identities between Noctuidae species. CSPs were broadly expressed in various organs, and SlituCSP13 was a DEG in adult antennae. The functional analysis in the Drosophila OR67d expression system found that SlituOR50, a receptor highly expressed in female antennae, is selectively tuned to farnesyl acetate. The results provide a solid foundation for understanding the molecular mechanisms by which chemosensory genes operate to elicit behavioral responses in polyphagous insects.

Nanoparticle vaccines based on the receptor binding domain of porcine deltacoronavirus elicit robust protective immune responses in mice.

Porcine deltacoronavirus (PDCoV), a novel swine enteropathogenic coronavirus, challenges the global swine industry. Currently, there are no approaches preventing swine from PDCoV infection. A new PDCoV strain named JS2211 was isolated. Next, the dimer receptor binding domain of PDCoV spike protein (RBD-dimer) was expressed using the prokaryotic expression system, and a novel nanoparticle containing RBD-dimer and ferritin (SC-Fe) was constructed using the SpyTag/SpyCatcher system. Finally, the immunoprotection of RBD-Fe nanoparticles was evaluated in mice. The novel PDCoV strain was located in the clade of the late Chinese isolate strains and close to the United States strains. The RBD-Fe nanoparticles were successfully established. Immune responses of the homologous prime-boost regime showed that RBD-Fe nanoparticles efficiently elicited specific humoral and cellular immune responses in mice. Notably, high level PDCoV RBD-specific IgG and neutralizing antibody (NA) could be detected, and the histopathological results showed that PDCoV infection was dramatically reduced in mice immunized with RBD-Fe nanoparticles. This study effectively developed a candidate nanoparticle with receptor binding domain of PDCoV spike protein that offers protection against PDCoV infection in mice.

Cell-free synthesis of the Salmonella specific broad host range bacteriophage, felixO1

Phage-based biocontrol of foodborne Salmonella is limited by the requisite use of Salmonella to propagate the phages. This limitation can be circumvented by producing Salmonella phages using a cell-free gene expression system (CFE) with a non-pathogenic chassis. Here, we produce the Salmonella phage felixO1 using an E. coli-based CFE system.

A novel nucleic acid linker for multi-gene expression enhances plant and animal synthetic biology.

The production of compact vectors for gene stacking is hindered by a lack of effective linkers. Here, we report that a 26-nt nucleic acid linker, NAL1, from the fungus Glarea lozoyensis and its truncated derivatives could connect two genes as a bicistron, enabling independent translation in a maize protoplast transient expression system and human 293 Tcells. The optimized 9-nt NAL10 linker was then used to connect four genes driven by a bidirectional promoter; this combination was successfully used to reconstruct the astaxanthin biosynthesis pathway in transgenic maize. The short and efficient nucleic acid linker NAL10 can be widely used in multi-gene expression and synthetic biology in animals and plants.

Episomal Vectors for Stable Production of Recombinant Proteins and Engineered Antibodies.

There is tremendous interest in the production of recombinant proteins, particularly bispecific antibodies and antibody-drug conjugates for research and therapeutic use. Here, we demonstrate a highly versatile plasmid system that allows the rapid generation of stable Expi293 cell pools by episomal retention of transfected DNA. By linking protein expression to puromycin resistance through an attenuated internal ribosome entry site, we achieve stable cell pools producing proteins of interest. In addition, split intein-split puromycin-mediated selection of two separate protein expression cassettes allows the stable production of bispecific antibody-like molecules or antibodies with distinct C-terminal heavy chain modifications, such as an antigen on one chain and a sortase tag on the other chain. We also use this novel expression system to generate stable Expi293 cell pools that secrete sortase A Δ59 variant Srt4M. Using these reagents, we prepared a site-specific drug-to-antibody ratio of 1 antibody-siRNA conjugate. We anticipate the simple, robust, and rapid stable protein expression systems described here being useful for a wide variety of applications.

"An L2 Education without Love is not Education at All": a phenomenographic study of undergraduate EFL students’ perceptions of pedagogical love

The role of pedagogical love in second language (L2) education has long remained under-explored due to dogmatic religious and cultural beliefs. There is insufficient scholarship on this construct at undergraduate levels. To bridge this gap, the present study employed a phenomenographic design to uncover Iranian undergraduate students’ perceptions of pedagogical love and its features, realizations, and determinant factors. It invited 22 undergraduate L2 learners to attend a semi-structured interview and complete a narrative frame. The results of content and thematic analysis attained through MAXQDA software (v. 20) revealed that the participants had different perceptions of pedagogical love. They considered it as a non-romantic intimacy and respect, deep care for students and their emotions, and pure love based on mutual trust. Furthermore, it was found that pedagogical love is essential for L2 education as characterized by a mutual trust and respect, kindness, care, bonding, intimacy, and forgiveness. Regarding its realizations, the results demonstrated that pedagogical love shows itself through intimacy, classroom engagement/participation, confidence, academic performance, and mutual care, respect, and responsibility. Moreover, it was identified that teachers’ emotional literacy, teachers’ pedagogical expertise, and positive classroom rapport facilitated the implementation of loving pedagogy at undergraduate level. Finally, the findings indicated that loving pedagogy practice was mostly precluded by strict religious beliefs, stigmatizing socio-cultural norms, emotion expression, fear, and traditional educational systems. Implications for L2 teachers and educators are discussed to augment their understanding of pedagogical love, as an opportunity to grasp the emotional side of language education.

A drug stabilizable GAL80ds for conditional control of gene expression via GAL4-UAS and CRISPR-Cas9 systems in Drosophila

The binary GAL4-UAS expression system has been widely used in Drosophila to achieve tissue-specific expression of genes. To further allow for simultaneous spatial and conditional control of gene expression in existing GAL4 expression lines backgrounds, temperature and chemical controllable GAL80 variants have been engineered. Here we add a new drug stabilizable GAL80ds variant, by fusing it to a low-background DHFR-22-DD. We first quantify both single (DD-GAL80) and double (DD-GAL80-DD) architectures and show varied background and activation levels. Next, we demonstrate the utility of GAL80dsDrosophila line to regulate a cell death gene ectopically, in a drug-dependent manner, by utilizing an existing tissue-specific GAL4 driver that regulates the expression of a cell death gene under a UAS. Finally, we showcase the usefulness of GAL80ds in tight drug-mediated regulation of a target gene, from an endogenous locus, by utilizing an existing tissue-specific GAL4 to drive the expression of a dead Cas9 variant fused to the transcriptional coactivator nejire, under a UAS and in gRNA lines. Overall, these new GAL80ds lines expand the use of the wide variety of existing tissue-specific GAL4 and gene-specific gRNA lines. This enables conditional control of genes, both ectopically and endogenously, for a broad array of gene expression control applications.

Thermophilic β-mannanases from bacteria: production, resources, structural features and bioengineering strategies.

β-mannanases are pivotal enzymes that cleave the mannan backbone to release short chain mannooligosaccharides, which have tremendous biotechnological applications including food/feed, prebiotics and biofuel production. Due to the high temperature conditions in many industrial applications, thermophilic mannanases seem to have great potential to overcome the thermal impediments. Thus, structural analysis of thermostable β-mannanases is extremely important, as it could open up new avenues for genetic engineering, and protein engineering of these enzymes with enhanced properties and catalytic efficiencies. Under this scope, the present review provides a state-of-the-art discussion on the thermophilic β-mannanases from bacterial origin, their production, engineering and structural characterization. It covers broad insights into various molecular biology techniques such as gene mutagenesis, heterologous gene expression, and protein engineering, that are employed to improve the catalytic efficiency and thermostability of bacterial mannanases for potential industrial applications. Further, the bottlenecks associated with mannanase production and process optimization are also discussed. Finally, future research related to bioengineering of mannanases with novel protein expression systems for commercial applications are also elaborated.

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System.

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

Characterization of two SGNH family cell death-inducing proteins from the horticulturally important fungal pathogen Botrytis cinerea based on the optimized prokaryotic expression system

Botrytis cinerea is one of the most destructive phytopathogenic fungi, causing significant losses to horticultural crops. As a necrotrophic fungus, B. cinerea obtains nutrients by killing host cells. Secreted cell death-inducing proteins (CDIPs) play a crucial role in necrotrophic infection; however, only a limited number have been reported. For high-throughput CDIP screening, we optimized the prokaryotic expression system and compared its efficiency with other commonly used protein expression systems. The optimized prokaryotic expression system showed superior effectiveness and efficiency and was selected for subsequent CDIP screening. The screening system verified fifty-five candidate proteins and identified two novel SGNH family CDIPs: BcRAE and BcFAT. BcRAE and BcFAT exhibited high expression levels throughout the infection process. Site-directed mutagenesis targeting conserved Ser residues abolished the cell death-inducing activity of both BcRAE and BcFAT. Moreover, the transient expression of BcRAE and BcFAT in plants enhanced plant resistance against B. cinerea without inducing cell death, independent of their enzymatic activities. Our results suggest a high-efficiency screening system for high-throughput CDIP screening and provide new targets for further study of B. cinerea-plant interactions.

Nav1.7 Modulator Bearing a 3-Hydroxyindole Backbone Holds the Potential to Reverse Neuropathic Pain.

Chronic pain is a growing global health problem affecting at least 10% of the world's population. However, current chronic pain treatments are inadequate. Voltage-gated sodium channels (Navs) play a pivotal role in regulating neuronal excitability and pain signal transmission and thus are main targets for nonopioid painkiller development, especially those preferentially expressed in dorsal root ganglial (DRG) neurons, such as Nav1.6, Nav1.7, and Nav1.8. In this study, we screened in virtual hits from dihydrobenzofuran and 3-hydroxyoxindole hybrid molecules against Navs via a veratridine (VTD)-based calcium imaging method. The results showed that one of the molecules, 3g, could inhibit VTD-induced neuronal activity significantly. Voltage clamp recordings demonstrated that 3g inhibited the total Na+ currents of DRG neurons in a concentration-dependent manner. Biophysical analysis revealed that 3g slowed the activation, meanwhile enhancing the inactivation of the Navs. Additionally, 3g use-dependently blocked Na+ currents. By combining with selective Nav inhibitors and a heterozygous expression system, we demonstrated that 3g preferentially inhibited the TTX-S Na+ currents, specifically the Nav1.7 current, other than the TTX-R Na+ currents. Molecular docking experiments implicated that 3g binds to a known allosteric site at the voltage-sensing domain IV(VSDIV) of Nav1.7. Finally, intrathecal injection of 3g significantly relieved mechanical pain behavior in the spared nerve injury (SNI) rat model, suggesting that 3g is a promising candidate for treating chronic pain.

Cytokines in influenza in children (literature review)

This literature review was compiled according to the data of search engines PubMed, eLIBRARY.RU, Google Scholar using key words influenza, cytokines, children. The article highlights the participation of pro-inflammatory and antiinflammatory cytokines in the pathogenesis of influenza, compares the cytokine profile in different strains of the virus, with concomitant pathology, as well as attempts to find an answer to the question: “Why are young children more susceptible to this infection?” The article contains information about the creation of express systems for diagnosing cytokine storm in patients with influenza and other acute respiratory viral infections, taking into account a complex of clinical and laboratory data that comes to the aid of a practicing physician. It should be noted that there are different opinions among authors regarding the early markers of severe and complicated influenza in childhood. This information makes the topic of studying the significance of cytokines in influenza in children relevant, but it requires continuation and clarification of knowledge on this issue.

Genetically encoded site-specific 19F unnatural amino acid incorporation in V. natriegens for in-cell NMR analysis

Nuclear magnetic resonance (NMR) spectroscopy NMR is a well-established technique for probing protein structure, dynamics and conformational changes. Taking advantage of the high signal sensitivity and broad chemical shift range of 19F nuclei, 19F NMR has been applied to investigate protein function at atomic resolution. In this report, we extend the unnatural amino acid site-specific incorporation into V. natriegens, an alternate protein expression system. The unnatural amino acid L-4-trifluoromethylphenylalanine (tfmF) was site-specifically introduced into the mitogen-activated protein kinase MEKK3 in V. natriegens using genetically encoded technology, which will be an extensive method for in-cell protein structure and dynamic investigation.

Accumulation mechanism of metabolite markers identified by machine learning between Qingyuan and Xiushui counties in Polygonatum cyrtonema Hua

Polygonatum cyrtonema Hua is a traditional Chinese medicinal plant acclaimed for its therapeutic potential in diabetes and various chronic diseases. Its rhizomes are the main functional parts rich in secondary metabolites, such as flavonoids and saponins. But their quality varies by region, posing challenges for industrial and medicinal application of P. cyrtonema. In this study, 482 metabolites were identified in P. cyrtonema rhizome from Qingyuan and Xiushui counties. Cluster analysis showed that samples between these two regions had distinct secondary metabolite profiles. Machine learning methods, specifically support vector machine-recursive feature elimination and random forest, were utilized to further identify metabolite markers including flavonoids, phenolic acids, and lignans. Comparative transcriptomics and weighted gene co-expression analysis were performed to uncover potential candidate genes including CHI, UGT1, and PcOMT10/11/12/13 associated with these compounds. Functional assays using tobacco transient expression system revealed that PcOMT10/11/12/13 indeed impacted metabolic fluxes of the phenylpropanoid pathway and phenylpropanoid-related metabolites such as chrysoeriol-6,8-di-C-glucoside, syringaresinol-4'-O-glucopyranosid, and 1-O-Sinapoyl-D-glucose. These findings identified metabolite markers between these two regions and provided valuable genetic insights for engineering the biosynthesis of these compounds.

Expression of an endo-type β-agarase AgaDcat in Pichia pastoris and its biochemical characterization

Neoagarooligosaccharides (NAOS), derived from the hydrolysis of agarose by β-agarases, have been found to have various biological activities. Herein, AgaDcat, originated from marine agar-degrading bacterium Zobellia galactanivorans, was for the first time successfully recombined and expressed in a eukaryotic (Pichia pastoris) system. Compared with other endo-β-agarase, AgaDcat has potential applications in producing neoagarotetraose (NA4) and neoagarohexaose (NA6) effectively because of its product specificity. The optimal temperature and pH of AgaDcat expressed in P. pastoris were 50 °C and 7.0, respectively. The maximum hydrolysis of agarose was achieved after 8h reaction at 45 °C and pH 7.0 using 750 U/g β-agarase. Through regulating hydrolysis conditions, NAOS with controllable degrees of polymerization (mainly NA4 and NA6) could be obtained. The Km value of AgaDcat expressed in P. pastoris was 1.377 mg/mL, which was significantly lower than that of AgaDcat expressed in Escherichia coli (6.358 mg/mL). Moreover, the melting temperature of AgaDcat expressed in P. pastoris (53.1 °C) was 4.5 °C higher than that of AgaDcat expressed in E. coli. AgaDcat expressed in P. pastoris exhibited higher thermal stability and substrate affinity than that expressed in E. coli, which could be ascribed to the glycosylation of proteins during translation modification period in the yeast expression system.

Conservation of shibire and RpII215 temperature-sensitive lethal mutations between Drosophila and Bactrocera tryoni.

The sterile insect technique can suppress and eliminate population outbreaks of the Australian horticultural pest, Bactrocera tryoni, the Queensland fruit fly. Sterile males mate with wild females that produce inviable embryos, causing population suppression or elimination. Current sterile insect releases are mixed sex, as the efficient removal of unrequired factory-reared females is not yet possible. In this paper, we assessed the known Drosophila melanogaster temperature-sensitive embryonic lethal alleles shibire (G268D, shits1) and RNA polymerase II 215 (R977C, RpII215ts) for potential use in developing B. tryoni genetic sexing strains (GSS) for the conditional removal of females. Complementation tests in D. melanogaster wild-type or temperature-sensitive genetic backgrounds were performed using the GAL4-UAS transgene expression system. A B. tryoni wild-type shibire isoform partially rescued Drosophila temperature lethality at 29°C by improving survivorship to pupation, while expressing B. tryoni shits1 failed to rescue the lethality, supporting a temperature-sensitive phenotype. Expression of the B. tryoni RpII215 wild-type protein rescued the lethality of D. melanogaster RpII215ts flies at 29°C. Overexpressing the B. tryoni RpII215ts allele in the D. melanogaster wild-type background unexpectedly produced a dominant lethal phenotype at 29°C. The B. tryoni shibire and RpII215 wild-type alleles were able to compensate, to varying degrees, for the function of the D. melanogaster temperature-sensitive proteins, supporting functional conservation across species. Shibire and RpII215 hold potential for developing insect strains that can selectively kill using elevated temperatures; however, alleles with milder effects than shits1 will need to be considered.