biostudies-arrayexpressAntonio RinaldiHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14355Our previous work has shown that exogenous DNA impose a burden to the host cells due to the finite amount of transcriptional and translational resources, resulting in a limiting productivity capacity. We thus designed context-aware genetic networks in which miRNAs were successfully exploited as burden mitigators, and observed upregulation of protein expression in the targeted cells. We co-transfected H1299, the testbed of our previous studies with a bi-directional plasmid encoding EGFP and mKate, either in an open loop circuit architecture (OL) that lack miRNA regulation, or in the form of a miRNA-iFFL, placing miR31 target sites, highly expressed in this cell line, in the 3’ (iFFL-3’) or 5’(iFFL-5’) UTR of mKate (Fig. 2a). We next sorted the cells that were either non-transfected (NTr) or transfected (Tr) according to fluorescence expression, to compare the OL that are more sensitive to burden, with iFFL-3’ and iFFL-5’ architectures.biostudies-arrayexpressLibrary Construction - Libraries were prepared using QuantSeq 3’ mRNA sequencing for RNA quantification kit (Lexogen)Sequencing - Samples were processed with NovaSeq 6000 System (Illumina).Sample Treatment - Transfections were carried out in 24-well plate for flow cytometry analysis. H1299 cells were transfected with Lipofectamine® 3000 according to manufacturer’s instructions and 300 ng of total DNA in 24-well plates. CHO and HEK293 were transfected with PEI transfection reagent with 300 ng of total DNA in 24-well plates. All cells were plated approximately 24h before transfection at 70000 to 80000 cells per well in 24-wells plates. At the moment of transfection, cells were put in starvation conditions, meaning in poor media for the first 24h. DNA was diluted in Opti-MEM reduced serum media (Gibco), before being mixed and incubated for 25 minutes prior to addition to the cells. All drugs were added 4h after transfection. Modified-RNAs (modRNA) were transfected using Lipofectamine® 3000 protocol under modified conditions in which no P3000 reagent was used. RNA was first diluted in Opti-MEM reduced serum media (Gibco) and it was then mixed with diluted Lipofectamine reagent as manufacturer’s instructions. The mix was incubated for 7 minutes prior to addition to the cells. The fast-forward protocol was used by seeding 120000 cells per well in 24-wells plates at the moment of transfection.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Sequence reads were trimmed using bbduk software to remove adapter sequences, poly-A tails and low-quality end bases. Alignment was performed with STAR on the Homo sapiens reference (hg38) provided by UCSC Genome Browser. The expression level of genes was determined with htseq-count using the Gencode/Ensembl gene modelMetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNAHomo sapiensAntonio RinaldifalseRNA-Seq of H1299 cells transfected with different types of bi-directional plasmid to better understand the transcriptional changes occurring with the different design frameworksOur previous work has shown that exogenous DNA impose a burden to the host cells due to the finite amount of transcriptional and translational resources, resulting in a limiting productivity capacity. We thus designed context-aware genetic networks in which miRNAs were successfully exploited as burden mitigators, and observed upregulation of protein expression in the targeted cells. We co-transfected H1299, the testbed of our previous studies with a bi-directional plasmid encoding EGFP and mKate, either in an open loop circuit architecture (OL) that lack miRNA regulation, or in the form of a miRNA-iFFL, placing miR31 target sites, highly expressed in this cell line, in the 3’ (iFFL-3’) or 5’(iFFL-5’) UTR of mKate (Fig. 2a). We next sorted the cells that were either non-transfected (NTr) or transfected (Tr) according to fluorescence expression, to compare the OL that are more sensitive to burden, with iFFL-3’ and iFFL-5’ architectures.2025-07-24T00:00:00Z2025-07-25T00:00:58.083Z2024-08-12T20:37:54.244ZE-MTAB-14355ERP163206EFO_0004170EFO_0003816EFO_0003738EFO_0004184EFO_0003969