Random sequence library in E. coli (Amplicon sequencing)
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ABSTRACT:
PROVIDER: PRJEB48999 | ENA |
REPOSITORIES: ENA
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Project description:Our ability to predict protein expression from DNA sequence alone remains poor, reflecting our limited understanding of cis-regulatory grammar and hampering the design of engineered genes for synthetic biology applications. Here, we generate a model that predicts the translational efficiency of the 5’ untranslated region (UTR) of mRNAs in the yeast Saccharomyces cerevisiae. We constructed a library of half a million 50-nucleotide- long random 5’ UTRs and assayed their activity in a massively parallel growth selection experiment. The resulting data allow us to quantify the impact on translation of Kozak sequence composition, upstream open reading frames (uORFs) and secondary structure. We trained a convolutional neural network (CNN) on the random library and showed that it performs well at predicting the translational efficiency of both a held-out set of the random 5’ UTRs as well as native S. cerevisiae 5’ UTRs. The model additionally was used to computationally evolve highly translating 5’ UTRs. We confirmed experimentally that the great majority of the evolved sequences lead to higher translation rates than the starting sequences, demonstrating the predictive power of this model. The sequences were assayed in the context of a constant promoter and downstream coding sequence. Cell growth was used as a proxy for protein expression. Growth was measured by determining the ratio of sequence counts before and after growth selection.
2017-09-27 | GSE104252 | GEO
Project description:Random sequences are an abundant source of bioactive RNAs or peptides
| PRJEB19640 | ENA
Project description: Not available
| S-EPMC4476321 | biostudies-literature
Project description:Peptides have great potential to combat antibiotic resistance. While many platforms can screen peptides for their ability to bind to target cells, there are virtually no platforms that directly assess the functionality of peptides. This limitation is exacerbated when identifying antimicrobial peptides, since the phenotype, death, selects against itself, and has caused a scientific bottleneck confining research to only a few naturally occurring classes of antimicrobial peptides. We have used this seeming dissonance to develop Surface Localized Antimicrobial displaY (SLAY); a platform that allows screening of unlimited numbers of peptides of any length, composition, and structure in a single tube for antimicrobial activity. Using SLAY, we screened ~800,000 random peptide sequences for antimicrobial function and identified thousands of active sequences doubling the number of known antimicrobial sequences. SLAY hits present with different potential mechanisms of peptide action and access to areas of antimicrobial physicochemical space beyond what nature has evolved.
2018-01-03 | GSE94530 | GEO
Project description:Peptides have great potential to combat antibiotic resistance. While many platforms can screen peptides for their ability to bind to target cells, there are virtually no platforms that directly assess the functionality of peptides. This limitation is exacerbated when identifying antimicrobial peptides, since the phenotype, death, selects against itself, and has caused a scientific bottleneck confining research to only a few naturally occurring classes of antimicrobial peptides. We have used this seeming dissonance to develop Surface Localized Antimicrobial displaY (SLAY); a platform that allows screening of unlimited numbers of peptides of any length, composition, and structure in a single tube for antimicrobial activity. Using SLAY, we screened ~800,000 random peptide sequences for antimicrobial function and identified thousands of active sequences doubling the number of known antimicrobial sequences. SLAY hits present with different potential mechanisms of peptide action and access to areas of antimicrobial physicochemical space beyond what nature has evolved.
2018-01-03 | GSE94529 | GEO
Project description:The half-life of mRNAs, as well as their translation, increases proportionally to the optimal codons, indicating a tight coupling of codon-dependent differential translation and degradation. Little is known about the regulation of this coupling. We found that the coupling in yeast displays a dichotomous behavior regarding the mRNA coding sequence length. Below a critical length, codon optimality fails to affect the stability of mRNAs even though they can be efficiently translated into short peptides and proteins. Above this threshold length, codon optimality-dependent differential mRNA stability emerges in a switch-like fashion, which coincides with a similar increase in the polysome propensity of the mRNAs. This threshold length can be tuned by the untranslated regions (UTR). Some of these UTRs can destabilize mRNAs without reducing translation, which plays a role in controlling the amplitude of the oscillatory expression of cell-cycle genes. Our findings help understand the translation of short peptides from noncoding RNAs and the translation by localized monosomes in neurons.
2023-09-28 | GSE235627 | GEO
Project description:The information about when and where each gene is to be expressed is mainly encoded in the DNA sequence of enhancers, sequence elements that comprise binding sites (motifs) for different transcription factors (TFs). Most of the research on enhancer sequences has been focused on TF motif presence, while the enhancer syntax, i.e. the flexibility of important motif positions and how the sequence context modulates the activity of TF motifs, remain poorly understood. Here, we explore the rules of enhancer syntax by a two-pronged approach in Drosophila melanogaster S2 cells: we (1) replace important motifs by an exhaustive set of all possible 65,536 eight-nucleotide-long random sequences and (2) paste eight important TF motif types into 763 motif positions within 496 enhancers. These complementary strategies reveal that enhancers display constrained sequence flexibility and the context-specific modulation of motif function. Important motifs can be functionally replaced by hundreds of sequences constituting several distinct motif types, but only a fraction of all possible sequences and motif types restore enhancer activity. Moreover, TF motifs contribute with different intrinsic strengths that are strongly modulated by the enhancer sequence context (the flanking sequence, presence and diversity of other motif types, and distance between motifs), such that not all motif types can work in all positions. Constrained sequence flexibility and the context-specific modulation of motif function are also hallmarks of human enhancers and TF motifs, as we demonstrate experimentally. Overall, these two general principles of enhancer sequences are important to understand and predict enhancer function during development, evolution and in disease.
2022-12-15 | GSE211655 | GEO
Project description:A computer program was used to create random amino acid sequences based on and restricted by physical shadow masks which will be used for lithography-based synthesis of peptides. The output from this algorithm was used to create peptides that were synthesized by Sigma Aldrich, and printed onto glass slides. The arrays contained 384 peptides printed in duplicate for each of 4 different mask designs. 52 different monoclonal antibodies were incubated on these microarrays and analyzed for their propensity to bind the peptides created from each mask set. The diversity of binding served as a proxy for the 'randomness' of these peptides, and provided information about how many masks are needed to truly generate random sequence peptides.
2014-01-16 | GSE49217 | GEO
Project description:Current spatial transcriptomic methods have been widely used to resolve gene expression; however, these methods are limited to fresh or fresh-frozen samples due to unsuccess of oligo(dT) primers in degraded RNA samples. Here we develop a spatial random-sequencing (spRandom-seq) technology for Formalin-fixed paraffin-embedded (FFPE) tissues by capturing full-length total RNAs with random primers. This approach provides a powerful spatial platform for clinical FFPE specimens and promises enormous applications in biomedicine.
2023-12-31 | GSE247881 | GEO
Project description: Not available
| S-EPMC6437963 | biostudies-literature