Gene Library Research Articles

Highly Efficient Synthesis of Rare Sugars from Glycerol in Endotoxin-Free ClearColi by Fermentation.

Rare sugars possess potential applications as low-calorie sweeteners, especially for anti-obesity and anti-diabetes. In this study, a fermentation biosystem based on the "DHAP-dependent aldolases strategy" was established for D-allulose and D-sorbose production from glycerol in endotoxin-free ClearColi BL21 (DE3). Several engineering strategies were adopted to enhance rare sugar production. Firstly, the combination of different plasmids for aldO, rhaD, and yqaB expression was optimized. Then, the artificially constructed ribosomal binding site (RBS) libraries of aldO, rhaD, and yqaB genes were assembled individually and combinatorially. In addition, a peroxidase was overexpressed to eliminate the damage or toxicity from hydrogen peroxide generated by alditol oxidase (AldO). Finally, stepwise improvements in rare sugar synthesis were elevated to 15.01 g/L with a high yield of 0.75 g/g glycerol in a 3 L fermenter. This research enables the effective production of rare sugars from raw glycerol in high yields.

Whole exome data prioritization unveils the hidden weight of Mendelian causes of male infertility. A report from the first Italian cohort.

Almost 40% of infertile men cases are classified as idiopathic when tested negative to the current diagnostic routine based on the screening of karyotype, Y chromosome microdeletions and CFTR mutations in men with azoospermia or oligozoospermia. Rare monogenic forms of infertility are not routinely evaluated. In this study we aim to investigate the unknown potential genetic causes in couples with pure male idiopathic infertility by applying variant prioritization to whole exome sequencing (WES) in a cohort of 99 idiopathic Italian patients. The ad-hoc manually curated gene library prioritizes genes already known to be associated with more common and rare syndromic and non-syndromic male infertility forms. Twelve monogenic cases (12.1%) were identified in the whole cohort of patients. Of these, three patients had variants related to mild androgen insensitivity syndrome, two in genes related to hypogonadotropic hypogonadism, and six in genes related to spermatogenic failure, while one patient is mutant in PKD1. These results suggest that NGS combined with our manually curated pipeline for variant prioritization and classification can uncover a considerable number of Mendelian causes of infertility even in a small cohort of patients.

Monitoring of a microbial community during bioaugmentation with hydrogenotrophic methanogens to improve methane yield of an anaerobic digestion process

Methane production by microbial fermentation of municipal waste is a challenge for better yield processes. This work describes the characterization of a hydrogenotrophic methanogen microbial community used in a bioaugmentation procedure to improve the methane yield in a thermophilic anaerobic process, digesting the organic fraction of municipal solid waste. The performance of the bioaugmentation was assessed in terms of methane production and changes in the microbial community structure. The results showed that bioaugmentation slightly improved the cumulative methane yield (+ 4%) in comparison to the control, and its use led to an acceleration of the methanogenesis stage. We observed associated significant changes in the relative abundance of taxa and their interactions, using high throughput DNA sequencing of V3-16S rRNA gene libraries, where the abundance of the archaeal hydrogenotrophic genus Methanoculleus (class Methanomicrobia, phylum Euryarchaeota) and the bacterial order MBA08 (class Clostridia, phylum Firmicutes) were dominant. The relevant predicted metabolic pathways agreed with substrate degradation and the anaerobic methanogenic process. The purpose of the study was to evaluate the effect of the addition of hydrogenotrophic methanogens in the generation of methane, while treating organic waste through anaerobic digestion.

Microbial Community Affects Daqu Quality and the Production of Ethanol and Flavor Compounds in Baijiu Fermentation.

Daqu is a traditional starter for Baijiu fermentation and is produced by spontaneous fermentation of ground and moistened barley or wheat. The quality of Daqu is traditionally evaluated based on physicochemical and subjective sensory parameters without microbiological analysis. Here, we compared the physicochemical characteristics of qualified (QD) and inferior (ID) Daqu, their microbial communities based on plate counting and PacBio SMRT sequencing of rRNA gene libraries, and their impacts on Baijiu fermentation. The results showed that the glucoamylase and α-amylase activities of QD were significantly higher than those of ID. The counts of yeasts and relative abundances of functional microbes, especially the amylolytic bacterium Bacillus licheniformis and fungi Saccharomycopsis fibuligera and Lichtheimia ramosa, were significantly higher in QD than in ID. The laboratory-scale Baijiu fermentation tests showed that the relative abundances of the amylolytic microbes were higher in the QD than the ID fermentation set, resulting in more efficient fermentation, as indicated by more weight loss and higher moisture content in the former. Consequently, more glycerol, acetic acid, ethanol, and other volatile compounds were produced in the QD than in the ID fermentation set. The results suggest that Daqu quality is determined by, and can be evaluated based on, its microbial community.

Optimal placement method of multi-objective and multi-type sensors for courtyard-style timber historical buildings based on Meta-genetic algorithm

Optimal sensor placement for timber architecture heritage poses a significant challenge due to the unique structural types and complex monitoring purposes. In this study, a three-stage method is proposed, taking a courtyard-style heritage, built 133 years ago, as an example. First, a finite element model that accounted for the parameter randomness and initial damage was constructed using a genetic algorithm (GA) and experimental results. Second, a new weighted fitness function of logarithmic type was developed for multi-type sensors and multi-objective monitoring. Third, a novel genetic algorithm, Meta-GA, was proposed, introducing competition group mechanisms and gene libraries to improve optimal capability while maintaining computational efficiency. The Meta-GA is then compared to the other two optimization modes using seven indexes. Finally, damage detection capability was tested for the proposed three schemes at noise levels of 0%, 5%, and 10%. The results reveal that the proposed three-stage method with Meta-GA can provide the best solution.

A long non-coding RNA essential for early embryonic development improves SCNT efficiency in goats.

Although abundant long non-coding RNAs (lncRNAs) have been found to be highly expressed in early embryos, the functions and mechanisms of most lncRNAs in regulating embryonic development remain unclear. This study was conducted to identify the key lncRNAs during embryonic genome activation (EGA) for promoting embryonic development after somatic cell nuclear transfer (SCNT) in goats. We screened and characterized lncRNAs from transcriptome data of in vitro-fertilized, 2-cell (IVF-2c) and 8-cell embryos (IVF-8c) and 8-cell SCNT embryos (SCNT-8c). We obtained 12 differentially expressed lncRNAs that were highly expressed in IVF-8c embryos compared to IVF-2c, and less expressed in SCNT-8c embryos. After target gene prediction, expression verification, and functional deletion experiments, we found that the expression level of lncRNA3720 affected the early embryonic development in goats. We cloned full length lncRNA3720 and over-expressed it in goat fetal fibroblasts (GFFs). We identified histone variants by analyzing the transcriptome data from both GFFs and embryos. Gene annotation of the gene library and literature search revealed that histone variants may have important roles in early embryo development, so we selected them as the potential target genes for lncRNA3720. Lastly, we compensated for the low expression of lncRNA3720 in SCNT embryos by microinjection, and showed that the development rate and quality of SCNT embryos was significantly improved. We speculate that lncRNA3720 is a key promoter of embryonic development in goats by interacting with histone variants.

LncRNA activates immune response against Vibrio anguillarum in the intestine-liver axis of turbot (Scophthalmus maximus L.) by sponging miRNA in a ceRNA regulatory network

Vibriosis, most commonly caused by the bacterium Vibrio anguillarum, is one of the most important diseases affecting the marine aquaculture industry throughout the world. The intestine is the main entry portal of V. anguillarum into host fishes. Although there has been extensive research on the immunity of the gut-associated lymphoid tissue (GALT) in teleost, the mechanisms underlying the immunity of the intestine-liver during bacterial infections are still not well understood. This study conducted an integrative analysis of the transcriptome in turbot (Scopthalmus maximus L.) intestine and liver after V. anguillarum infection. To provide evidence of immunological collaboration in the turbot intestine-liver axis, we constructed the immune gene libraries, and conducted the functional annotation and enrichment analyses of immune genes that were jointly expressed in both turbot intestine and liver. Additionally, we administered the bioinformatic analyses of transcriptomic data to investigate regulatory relationships between non-coding RNAs (ncRNAs) and their target immune genes, based on the ceRNA theory. The recognition and binding abilities to bacteria were activated by the Toll-like receptor (TLR) signaling pathway. Subsequently, NF-κB was activated by the TLR/MyD88 signaling, and its target genes were induced to up-regulate in intestine and associated tissues to the digestive tract to enhance disease resistance through the production of inflammation. Histology of the intestine and liver of infected fish showed pathological changes consistent with inflammation. Bioinformatic analysis suggested that ceRNA regulatory networks play a pivotal role in the regulation of TLR genes, and RT-qPCR analyses and dual-luciferase reporter assays showed confirmed evidence to verify these hypothesized regulatory relationships. Overall, the results in this study demonstrated that a lncRNA (LNC_002468) activated the TLR signaling pathway by upregulating the TLR9 gene through sponging miRNA (novel_709). This study provides a strong indication that ncRNAs play regulatory roles in the antibacterial immune response of teleost fishes. However, further functional verification is required of the key immune genes and their regulatory relationship with miRNAs and lncRNAs.

Characterization of Two Na+(K+, Li+)/H+ Antiporters from Natronorubrum daqingense

The Na+/H+ antiporter NhaC family protein is a kind of Na+/H+ exchanger from the ion transporter (IT) superfamily, which has mainly been identified in the halophilic bacteria of Bacillus. However, little is known about the Na+/H+ antiporter NhaC family of proteins in the extremely halophilic archaea. In this study, two Na+/H+ antiporter genes, nhaC1 and nhaC2, were screened from the genome of Natronorubrum daqingense based on the gene library and complementation of salt-sensitive Escherichia coli KNabc. A clone vector pUC18 containing nhaC1 or nhaC2 could make KNabc tolerate 0.6 M/0.7 M NaCl or 30 mM/40 mM LiCl and a pH of up to 8.5/9.5, respectively. Functional analysis shows that the Na+(K+, Li+)/H+ antiport activities of NhaC1 and NhaC2 are both pH-dependent in the range of pH 7.0–10.0, and the optimal pH is 9.5. Phylogenetic analysis shows that both NhaC1 and NhaC2 belong to the Na+/H+ antiporter NhaC family of proteins and are significantly distant from the identified NhaC proteins from Bacillus. In summary, we have identified two Na+(K+, Li+)/H+ antiporters from N. daqingense.

Bacterial Community Shifts during Polyp Bail-Out Induction in Pocillopora Corals.

Polyp bail-out constitutes both a stress response and an asexual reproductive strategy that potentially facilitates dispersal of some scleractinian corals, including several dominant reef-building taxa in the family Pocilloporidae. Recent studies have proposed that microorganisms may be involved in onset and progression of polyp bail-out. However, changes in the coral microbiome during polyp bail-out have not been investigated. In this study, we induced polyp bail-out in Pocillopora corals using hypersaline and hyperthermal methods. Bacterial community dynamics during bail-out induction were examined using the V5-V6 region of the 16S-rRNA gene. From 70 16S-rRNA gene libraries constructed from coral tissues, 1,980 OTUs were identified. Gammaproteobacteria and Alphaproteobacteria consistently constituted the dominant bacterial taxa in all coral tissue samples. Onset of polyp bail-out was characterized by increased relative abundance of Alphaproteobacteria and decreased abundance of Gammaproteobacteria in both induction experiments, with the shift being more prominent in response to elevated temperature than to elevated salinity. Four OTUs, affiliated with Thalassospira, Marisediminitalea, Rhodobacteraceae, and Myxococcales, showed concurrent abundance increases at the onset of polyp bail-out in both experiments, suggesting potential microbial causes of this coral stress response. IMPORTANCE Polyp bail-out represents both a stress response and an asexual reproductive strategy with significant implications for reshaping tropical coral reefs in response to global climate change. Although earlier studies have suggested that coral-associated microbiomes likely contribute to initiation of polyp bail-out in scleractinian corals, there have been no studies of coral microbiome shifts during polyp bail-out. In this study, we present the first investigation of changes in bacterial symbionts during two experiments in which polyp bail-out was induced by different environmental stressors. These results provide a background of coral microbiome dynamics during polyp bail-out development. Increases in abundance of Thalassospira, Marisediminitalea, Rhodobacteraceae, and Myxococcales that occurred in both experiments suggest that these bacteria are potential microbial causes of polyp bail-out, shedding light on the proximal triggering mechanism of this coral stress response.

Prevalence of Deltaproteobacterial sequences in nifH gene pools associated with the rhizosphere of native switchgrass from Tall Grass Prairie (Oklahoma, USA).

The potential nitrogen-fixing bacterial diversity in the rhizospheric soil of the native switchgrass (Panicum virgatum L.) from Tall Grass Prairies of Northern Oklahoma was studied using a partial region of nitrogenase structural gene-nifH. Eleven clone libraries constructed from nifH amplicons gave 407 good-quality sequences. More than 70% of sequences showed similarity of nifH with uncultured bacteria (< 98%). The dominance of sequences affiliated with Deltaproteobacterial nifH was observed, followed by Betaproteobacterial nifH sequences. The nifH gene library was dominated by the genera Geobacter, Rhizobacter, Paenibacillus, and Azoarcus. Sequences affiliated with rhizobia, such as Bradyrhizobium, Methylocystis, Ensifer, etc., were also in the rhizosphere in small numbers. From Deltaproteobacteria, five genera, namely Geobacter, Pelobacter, Geomonas, Desulfovibrio, and Anaeromyxobacter, contributed to 48% of the total sequences suggesting the dominance of group Deltaproteobacteria in the rhizosphere of native switchgrass. Considering the percent similarity of the nifH sequences with cultivated bacteria, this study demonstrated the presence of novel bacterial species in switchgrass rhizospheric soil from Tall Grass Prairie.

Genome Sequence Resource of Curvularia clavata Causing Leaf Spot Disease on Tobacco by Oxford Nanopore PromethION.

Genome Sequence Resource of Curvularia clavata Causing Leaf Spot Disease on Tobacco by Oxford Nanopore PromethION.

MALDI-MS screening of microbial colonies with isomer resolution to select fatty acid desaturase variants

Creating controlled lipid unsaturation locations in oleochemicals can be a key to many bioengineered products. However, evaluating the effects of modifications to the acyl-ACP desaturase on lipid unsaturation is not currently amenable to high-throughput assays, limiting the scale of redesign efforts to <200 variants. Here, we report a rapid MS assay for profiling the positions of double bonds on membrane lipids produced by Escherichia coli colonies after treatment with ozone gas. By MS measurement of the ozonolysis products of Δ6 and Δ8 isomers of membrane lipids from colonies expressing recombinant Thunbergia alata desaturase, we screened a randomly mutagenized library of the desaturase gene at 5 s per sample. Two variants with altered regiospecificity were isolated, indicated by an increase in 16:1 Δ8 proportion. We also demonstrated the ability of these desaturase variants to influence the membrane composition and fatty acid distribution of E. coli strains deficient in the native acyl-ACP desaturase gene, fabA. Finally, we used the fabA deficient chassis to concomitantly express a non-native acyl-ACP desaturase and a medium-chain thioesterase from Umbellularia californica, demonstrating production of only saturated free fatty acids.

De novo transcriptomes of six calanoid copepods (Crustacea): a resource for the discovery of novel genes

This study presents eight new high-quality de novo transcriptomes from six co-occurring species of calanoid copepods, the first published for Neocalanus plumchrus, N. cristatus, Eucalanus bungii and Metridia pacifica and additional ones for N. flemingeri and Calanus marshallae. They are ecologically-important members of sub-arctic North Pacific marine zooplankton communities. ‘Omics data for this diverse and numerous taxonomic group are sparse and difficult to obtain. Total RNA from single individuals was used to construct gene libraries that were sequenced on an Illumina Next-Seq platform. Quality filtered reads were assembled with Trinity software and validated using multiple criteria. The study’s primary purpose is to provide a resource for gene expression studies. The integrated database can be used for quantitative inter- and intra-species comparisons of gene expression patterns across biological processes. An example of an additional use is provided for discovering novel and evolutionarily-significant proteins within the Calanoida. A workflow was designed to find and characterize unannotated transcripts with homologies across de novo assemblies that have also been shown to be eco-responsive.

A ubiquitous gammaproteobacterial clade dominates expression of sulfur oxidation genes across the mesopelagic ocean.

The deep ocean (>200 m depth) is the largest habitat on Earth. Recent evidence suggests sulfur oxidation could be a major energy source for deep ocean microbes. However, the global relevance and the identity of the major players in sulfur oxidation in the oxygenated deep-water column remain elusive. Here we combined single-cell genomics, community metagenomics, metatranscriptomics and single-cell activity measurements on samples collected beneath the Ross Ice Shelf in Antarctica to characterize a ubiquitous mixotrophic bacterial group (UBA868) that dominates expression of RuBisCO genes and of key sulfur oxidation genes. Further analyses of the gene libraries from the 'Tara Oceans' and 'Malaspina' expeditions confirmed the ubiquitous distribution and global relevance of this enigmatic group in the expression of sulfur oxidation and dissolved inorganic carbon fixation genes across the global mesopelagic ocean. Our study also underscores the unrecognized importance of mixotrophic microbes in the biogeochemical cycles of the deep ocean.

An Agrobacterium-Mediated Transient Expression Method for Functional Assay of Genes Promoting Disease in Monocots.

Agrobacterium-mediated transient expression (AMTE) has been widely used for high-throughput assays of gene function in diverse plant species. However, its application in monocots is still limited due to low expression efficiency. Here, by using histochemical staining and a quantitative fluorescence assay of β-glucuronidase (GUS) gene expression, we investigated factors affecting the efficiency of AMTE on intact barley plants. We found prominent variation in GUS expression levels across diverse vectors commonly used for stable transformation and that the vector pCBEP produced the highest expression. Additionally, concurrent treatments of plants with one day of high humidity and two days of darkness following agro-infiltration also significantly increased GUS expression efficiency. We thus established an optimized method for efficient AMTE on barley and further demonstrated its efficiency on wheat and rice plants. We showed that this approach could produce enough proteins suitable for split-luciferase assays of protein-protein interactions on barley leaves. Moreover, we incorporated the AMTE protocol into the functional dissection of a complex biological process such as plant disease. Based on our previous research, we used the pCBEP vector to construct a full-length cDNA library of genes upregulated during the early stage of rice blast disease. A subsequent screen of the library by AMTE identified 15 candidate genes (out of ~2000 clones) promoting blast disease on barley plants. Four identified genes encode chloroplast-related proteins: OsNYC3, OsNUDX21, OsMRS2-9, and OsAk2. These genes were induced during rice blast disease; however, constitutive overexpression of these genes conferred enhanced disease susceptibility to Colletotrichum higginsianum in Arabidopsis. These observations highlight the power of the optimized AMTE approach on monocots as an effective tool for facilitating functional assays of genes mediating complex processes such as plant-microbe interactions.

The mature phyllosphere microbiome of grapevine is associated with resistance against Plasmopara viticola.

Phyllosphere microbiota represents a substantial but hardly explored reservoir for disease resistance mechanisms. The goal of our study was to understand the link between grapevine cultivars susceptibility to Plasmopara viticola, one of the most devastating leaf pathogens in viticulture, and the phyllosphere microbiota. Therefore, we analyzed a 16S rRNA gene library for the dominant phyllosphere bacterial phyla Alphaproteobacteria of seven Vitis genotypes at different developmental stages, i.e., flowering and harvesting, via amplicon sequencing. Young leaves had significantly higher Alphaproteobacterial richness and diversity without significant host-specificity. In contrast, the microbial communities of mature leaves were structurally distinct in accordance with P. viticola resistance levels. This statistically significant link between mature bacterial phyllosphere communities and resistant phenotypes was corroborated by beta diversity metrics and network analysis. Beyond direct host-driven effects via the provision of microhabitats, we found evidence that plants recruit for specific bacterial taxa that were likely playing a fundamental role in mediating microbe-microbe interactions and structuring clusters within mature communities. Our results on grape-microbiota interaction provide insights for targeted biocontrol and breeding strategies.

Biogeographic distribution, ecotype partitioning and controlling factors of Chloroflexi in the sediments of six hadal trenches of the Pacific Ocean

The hadal trenches are “hot spots” for mineralization of organic matter in the deep ocean. Chloroflexi are one of the most dominant and active taxa in trench sediments, serving as important drivers of carbon cycles in hadal trenches. However, current understanding on hadal Chloroflexi is largely restricted to individual trench. This study systematically analyzed the diversity, biogeographic distribution, ecotype partitioning as well as environmental drivers of Chloroflexi in the sediments of hadal trenches, by reanalyzing 16S rRNA gene libraries of 372 samples from 6 trenches around the Pacific Ocean. The results showed that Chloroflexi averagely account for 10.10 % and up to 59.95 % of total microbial communities in the trench sediments. Positive correlations between relative abundance of Chloroflexi and depths down the vertical sediment profiles were observed in all of the sediment cores analyzed, suggesting the increasing significance of Chloroflexi in deeper sediment layers. Overall, trench sediment Chloroflexi were mainly composed of the classes Dehalococcidia, Anaerolineae and JG30-KF-CM66, and four orders i.e. SAR202, Anaerolineales, norank JG30-KF-CM66 and S085, were identified as core taxa that were dominant and prevalent in the hadal trench sediments. A total of 22 subclusters were identified within these core orders, and distinct patterns of ecotype partitioning related with depths down the vertical sediment profiles were observed, suggesting the great diversification of metabolic potentials and environment preference of different Chloroflexi lineages. The spatial distribution of hadal Chloroflexi were found to be significantly related with multiple environmental factors, while depths down the vertical sediment profiles explained the highest proportion of variations. These results provide valuable information for further exploring the roles of Chloroflexi in biogeochemical cycle of the hadal zone, and lay the foundation for understanding the adaptive mechanisms and evolutionary characteristics of microorganisms in hadal trenches.

Taxonomic Profiling of Fecal and Intestinal Microbiota in the African Turquoise Killifish Nothobranchius furzeri.

Killifish are emerging as a new laboratory system in which to study a range of questions, from the genetic basis of embryo dormancy to life history trait evolution, age-dependent neurodegeneration, and the connection between microbial community structure and biology of aging. Over the past decade, advances in high-throughput sequencing have helped uncover the vast diversity of microbial communities present in environmental samples and on host epithelia. Here, we describe an optimized protocol to study the taxonomic composition of intestinal and fecal microbiota in laboratory-raised as well as natural killifish populations and provide comprehensive step-by-step instructions for tissue sampling, high-throughput genomic DNA extraction, and the generation of 16S V3V4 rRNA and 16S V4 rRNA gene libraries.

Abstract 3471: A function-based high-throughput discovery platform, myeloid iOTarg, identifies novel immune checkpoints of the tumor microenvironment

Abstract Tumor-associated macrophages (TAMs) are important players to maintain immunosuppression within the tumor microenvironment (TME) and attenuate the function of effector immune cells to promote tumor survival. High density of TAMs is well-recognized as a feature of tumor progression and poor prognostic factor across various tumor types. Therefore, therapeutic strategies to dampen TAMs’ suppressive potential in TME and reprogram them towards a pro-inflammatory phenotype are being increasingly appreciated as being paramount for an effective immunotherapy. Nevertheless, the key mediators of TAM-induced immunosuppression remain largely unknown, identification of which can lead to the development of novel immunotherapeutic strategies aimed at re-educating the immunosuppressive TAMs to an anti-tumor phenotype. To systematically interrogate the genes responsible for the immune-suppressive phenotype of TAMs, we developed a functional, high-throughput genetic screening platform (iOTarg™) based on human primary monocyte-derived M2-like macrophages. Using CRISPR, we knocked out a proprietary library of well-expressed and druggable genes (1400 genes) in two independent donor-derived M2-like macrophages and cocultured them with activated autologous effector T cells (TCs). We achieved nearly complete knockout (KO) efficacy over a range of control genes. Functional impact of individual KO on macrophage viability and phenotype, as well as on TC activity, was measured using multiparametric assay readouts. Initial hits were reconfirmed in three additional donors in a secondary screen that integrated an additional tumor lysis readout. As expected, knockout of CSF1R, which is essential for macrophage maintenance, resulted in a dramatic loss of macrophage cell viability, whereas KO of established TAM markers, TREM2 and Clever-1, induced a change in macrophage phenotype. Furthermore, KO of immune-inhibitory receptor LILRB2 reduced the M2-like phenotype and restored TC activity. Inhibition of M2 activation of macrophages and subsequent increase in TC activation culminated in strong tumor cell killing for a subset of genes, highlighting an untapped repertoire of novel TAM-associated immune-checkpoint targets. Taken together, we report for the first time a highly sophisticated target discovery platform that addresses the functional role of any TAM-expressed gene in regulating macrophage viability, phenotype, T cell activity and even its gross impact on tumor cell lysis, all done in a high-throughput format employing CRISPR-edited primary human immune cells and multi-parametric functional immunological assays. As a result, we could confirm well-known targets in clinical testing, as well as identify additional novel targets that could lead to first-in-class, TME-based therapeutics and expansion of treatment options in immune oncology. Citation Format: Kritika Sudan, Tillmann Michels, Carmen Amerhauser, Claudia Tschulik, Leonie Majunke, Lucille Albert, Valentina Volpin, Adriana Turqueti-Neves, Ronny Milde, Nisit Khandelwal. A function-based high-throughput discovery platform, myeloid iOTarg, identifies novel immune checkpoints of the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3471.

Clonal Amplification-Enhanced Gene Expression in Synthetic Vesicles

In cell-free gene expression, low input DNA concentration severely limits the phenotypic output, which may impair in vitro protein evolution efforts. We address this challenge by developing CADGE, a strategy that is based on clonal isothermal amplification of a linear gene-encoding dsDNA template by the minimal Φ29 replication machinery and in situ transcription-translation. We demonstrate the utility of CADGE in bulk and in clonal liposome microcompartments to boost up the phenotypic output of soluble and membrane-associated proteins, as well as to facilitate the recovery of encapsulated DNA. Moreover, we report that CADGE enables the enrichment of a DNA variant from a mock gene library via either a positive feedback loop-based selection or high-throughput screening. This new biological tool can be implemented for cell-free protein engineering and the construction of a synthetic cell.