Engineering orthogonal signaling pathways reveals the sparse distribtion of protein protein interactions in sequence space [RNA-seq]
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PROVIDER: PRJNA528279 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8757050_1.fastq.gz | Fastqsanger.gz | |||
| SRR8757050_2.fastq.gz | Fastqsanger.gz | |||
| SRR8757060_1.fastq.gz | Fastqsanger.gz | |||
| SRR8757060_2.fastq.gz | Fastqsanger.gz | |||
| SRR8757041_1.fastq.gz | Fastqsanger.gz |
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Project description:Engineering orthogonal signaling pathways reveals the sparse distribtion of protein protein interactions in sequence space
| PRJNA494538 | ENA
Project description:Engineering orthogonal signaling pathways reveals the sparse distribtion of protein protein interactions in sequence space [degenerate_library_sort_seq]
| PRJNA494534 | ENA
Project description:Engineering orthogonal signaling pathways reveals the sparse distribtion of protein protein interactions in sequence space [combinatorial_library_sort_seq]
| PRJNA494535 | ENA
Project description:The specificity of the kinase-regulator interaction is driven by a limited set of interfacial residues in each protein that strongly. To identify combinations of these interface residues that are functional and potentially insulated from existing two-component signaling pathways in E. coli, we constructed a dual library of mutants in which the key, coevolving interface residues of a canonical two-component system, PhoQ and PhoP, were randomized. We used NNS codons to randomize six residues in PhoQ and five residues in PhoP, all of which lie at the interface formed by the two proteins in complex and that are critical to determining partner specificity in all two-component signaling pathways. To identify functional combinations of residues, we first grew the library of PhoQ-PhoP variants overnight in medium with low Mg2+, which activates PhoQ. Because cells must phosphorylate PhoP to grow when extracellular Mg2+ is limiting, this step enriches for functional PhoQ-PhoP variants. Variants that survived selection in limiting Mg2+ were then subjected to Sort-Seq, using fluorescence-activated cell sorting (FACS) and deep sequencing to quantify the signal responsiveness of variants in the library. To gauge the phosphorylation of PhoP in vivo, we used a fluorescent transcriptional reporter, PmgrB-yfp. In the presence of low extracellular Mg2+, functional PhoQ promotes the phosphorylation of PhoP and the production of YFP, whereas in the presence of high concentrations of Mg2+, PhoQ drives the dephosphorylation of PhoP, limiting the accumulation of YFP). The library was grown in each condition for 6 hours before sorting and sequencing. To identify variants that are signal responsive and drive YFP production specifically in low Mg2+, we sorted cells from each condition into 8 separate bins and deep sequenced the randomized regions of variants collected in each bin. We then calculated the frequency of each variant in each bin to yield the distributions of individual variants in low and high Mg2+, which were fit to Gaussians. From these fits, we assessed the mean level of YFP in each condition and the fold-induction, or signal responsiveness, of each variant detected in the library. The 11 codons / amino acids listed in this dataset refer to codons 12, 14, 15, 18, and 19 in PhoP and codons 284, 288, 289, 292, 302, and 303 in PhoQ, in that order.
2019-09-20 | GSE120780 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below.
2019-08-05 | GSE120789 | GEO
Project description:The cross compatibility of functional PhoQ-PhoP mutants with altered specificity residues was analyzed by sort seq. We cloned and combinatorially combined 79 PhoQ* and 71 PhoP* variants, producing a library with a theoretical diversity of 5,609. The library was then subjected to Sort-Seq, using fluorescence-activated cell sorting (FACS) and deep sequencing to quantify the signal responsiveness of variants in the library. To gauge the phosphorylation of PhoP in vivo, we used a fluorescent transcriptional reporter, PmgrB-yfp. In the presence of low extracellular Mg2+, functional PhoQ promotes the phosphorylation of PhoP and the production of YFP, whereas in the presence of high concentrations of Mg2+, PhoQ drives the dephosphorylation of PhoP, limiting the accumulation of YFP). The library was grown in each condition for 6 hours before sorting and sequencing. To identify variants that are signal responsive and drive YFP production specifically in low Mg2+, we sorted cells from each condition into 8 separate bins and deep sequenced the randomized regions of variants collected in each bin (Fig. 2b). We then calculated the frequency of each variant in each bin to yield the distributions of individual variants in low and high Mg2+, which were fit to Gaussians. From these fits, we assessed the mean level of YFP in each condition and the fold-induction, or signal responsiveness, of each variant detected in the library. The 11 codons / amino acids listed in this dataset refer to codons 12, 14, 15, 18, and 19 in PhoP and codons 284, 288, 289, 292, 302, and 303 in PhoQ, in that order.
2019-07-27 | GSE120786 | GEO
Project description:To test the global insulation of selected PhoQ*-PhoP* variants, we used RNA-Seq to examine gene expression in strains carrying one of six different PhoQ-PhoP variant pairs. In each case, cells were grown in medium with either excess or limiting extracellular Mg2+ to repress or stimulate PhoQ, respectively, before harvesting RNA. Each system produced a similar induction of known PhoP-dependent genes. To assess whether a variant PhoQ cross-phosphorylated other response regulators, we took advantage of the fact that, when active, most response regulators autoregulate and promote expression of themselves and their cognate histidine kinase2. Notably, none of the six strains tested showed significant induction of other two-component systems relative to a wild-type control. Similar results are seen for the chimeric pathway AQ4-PhoP4, which was induced with its ligand: trans-zeatin.
2019-09-20 | GSE128611 | GEO
Project description:Gene duplication is a common and powerful mechanism by which cells create new signalling pathways1,2, but recently duplicated proteins typically must become insulated from each other and from other paralogues to prevent unwanted crosstalk3. A similar challenge arises when new sensors or synthetic signalling pathways are engineered within cells or transferred between genomes. How easily new pathways can be introduced into cells depends on the density and distribution of paralogous pathways in the sequence space that is defined by their specificity-determining residues4,5. Here we directly investigate how crowded this sequence space is, by generating novel two-component signalling proteins in Escherichia coli using cell sorting coupled to deep sequencing to analyse large libraries designed on the basis of coevolutionary patterns. We produce 58 insulated pathways comprising functional kinase-substrate pairs that have different specificities than their parent proteins, and demonstrate that several of these new pairs are orthogonal to all 27 paralogous pathways in E. coli. Additionally, from the kinase-substrate pairs generated, we identify sets consisting of six pairs that are mutually orthogonal to each other, which considerably increases the two-component signalling capacity of E. coli. These results indicate that sequence space is not densely occupied. The relative sparsity of paralogues in sequence space suggests that new insulated pathways can arise easily during evolution, or be designed de novo. We demonstrate the latter by engineering a signalling pathway in E. coli that responds to a plant cytokinin, without crosstalk to extant pathways. Our work also demonstrates how coevolution-guided mutagenesis and the mapping of sequence space can be used to design large sets of orthogonal protein-protein interactions.
| S-EPMC6858568 | biostudies-literature
Project description:The Bxb1 bacteriophage serine DNA recombinase is an efficient tool for engineering recombinant DNA into the genomes of cultured cells. Generally, a single engineered “landing pad” site is introduced into the cell genome, permitting the integration of transgenic circuits or libraries of transgene variants. While sufficient for many studies, the extent of genetic manipulation possible with a single recombinase site is limiting, and insufficient for more complex cell-based assays for protein function. Here, we harnessed two orthogonal Bxb1 recombinase sites to enable new avenues for mammalian synthetic biology. By designing plasmids with two recombinase sites, we demonstrate that we can avoid genomic integration of undesirable bacterial DNA elements. We also created “double landing pad” cells simultaneously harboring two orthogonal Bxb1 recombinase sites. These cells allow transgenic protein variant libraries to be readily paired with assay-specific protein partners or biosensors, opening up new functional readouts for large-scale functional assays.
2023-05-05 | GSE231138 | GEO
Project description:Biomarkers unique for distributed stem cells (DSCs) have proven elusive. Previous searches for proteins expressed specifically in DSCs were hampered by difficulty obtaining pure DSCs and challenges to successfully mining complex molecular expression data. To identify novel candidates for DSC biomarkers, we combined a sparse feature selection method with combinatorial molecular expression data focused on asymmetric self-renewal, a defining DSC property. Our analyses revealed reduced expression of the histone H2A variant H2A.Z as a superior discriminator for asymmetric self-renewal, which proved to be a novel pattern-specific biomarker of DSCs. Overview: Sixteen cDNA micro-array samples were analyzed, with four replicates for each of four biologically orthogonal comparisons of congruent asymmetrically self-renewing versus symmetrically self-renewing cells. Sparse feature selection was used to identify a uniquely robust discriminator of these two different DSC self-renewal patterns. General Approach: An orthogonal-intersection strategy for identification of asymmetric self-renewal associated genes Previously, we used non-tumorigenic, immortalized mouse mammary epithelial C127 cells and mouse embryo fibroblasts (MEFs) to derive lines with conditional self-renewal patterns. The self-renewal pattern of these cells can be reversibly switched between symmetric and asymmetric by varying either culture temperature or Zn concentration, respectively, as a consequence of controlling p53 expression from respectively responsive promoters (1-6). These and related cell lines were used to design a 2 x 4 orthogonal-intersection microarray analysis for the purpose of identifying genes whose expression consistently showed the same pattern of change between asymmetric self-renewal versus symmetric self-renewal. Four different pair-wise comparisons were developed in which a state of asymmetric self-renewal was compared to a congruent state of symmetric self-renewal. The first 3 comparisons were based on a difference in p53 expression, but each had a different biological context. For asymmetric versus symmetric, respectively, these comparisons were: 1) Zn-inducible p53 MEFs (Ind-8 cells) versus p53-null control MEFs (Con-3 cells), both cultured in Zn-supplemented medium (65 micromolar ZnCl2); 2) Zn-inducible p53 MEFs in Zn-supplemented medium versus in Zn-free medium (1,3-6); and 3) low temperature (32.5?C)-inducible p53 mouse mammary epithelial cells (MMECs; 1h-3 cells) versus vector-control MMECs (i.e., no p53 transgene; 1g-1 cells), both cultured at 32.5?C (2,5,6). The fourth comparison had a special purpose. It provided a comparison of asymmetric versus symmetric self-renewal that was not due to a difference in p53 expression (1). Two previously described derivatives of the Zn-responsive p53-inducible MEFs were used to make this comparison. One line (tI-3 cells) is stably transfected with a constitutively expressed type II inosine monophosphate dehydrogenase (IMPDH II) gene (1,3,5). IMPDH II is the rate-limiting enzyme for guanine ribonucleotide biosynthesis. Its down-regulation by p53 is required for asymmetric self-renewal (3,5). Therefore, even in Zn-supplemented medium, which induces normal p53 expression, cells derived with a stably expressed IMPDH II transgene continue to undergo symmetric self-renewal (3,5). This abrogation of p53 effects on self-renewal pattern occurs even though other p53-dependent responses remain intact (3). Under the same conditions, control vector-only transfectants (tC-2 cells) continue to exhibit asymmetric self-renewal (3,5). Thus, this fourth comparison could be used to exclude genes whose change in expression was primarily due to changes p53 expression and not specifically transitions in self-renewal symmetry as well (1). We identified genes whose expression varied consistently for all 4 orthogonal comparisons of cells in states of asymmetric self-renewal versus symmetric self-renewal (7). We developed a new quality control metric called the population division cycle ratio (PDC ratio) to insure consistent degrees of asymmetric self-renewal and symmetric self-renewal across all 4 orthogonal comparisons (6,7). Prefabricated cDNA microarrays constructed with the National Institute for Aging (NIA) mouse 15K mouse clone set were used for the analysis (8,9). As detailed in supplemental Materials and Methods (see below), for each of the 4 experimental comparisons, we isolated two independent samples of RNA; and each of the RNA samples was labeled independently with Cy5 and Cy3 fluorescent dyes. Each independent set of fluorescent RNA samples was used to develop two reciprocal co-hybridizations for each experimental comparison. Thus, data from 4 microarrays, representing 2 independent experiments, were available for each of the 4 orthogonal contexts for asymmetric versus symmetric self-renewal. Materials and Methods: cDNA Microarray Data Analysis Cells for total RNA extraction were harvested at the time estimated for PDC = 2 (36 hours for Ind-8 and Con-3, 48 hours for tC-2, tI-3, 1h-3, and 1g-1), and were selected for microarray experiments based on the actual PDC ratio curves derived from replicate cell cultures grown in parallel. The same PDC ratio values were maintained to obtain similar fractions of cycling stem-like cells and non-cycling differentiating cells for all compared asymmetric models. Total RNA was extracted with the Trizol reagent (Invitrogen, Carlsbad, CA) and impurities were removed with the Qiagen RNeasy kit (Qiagen, Valencia, CA). Fifty to seventy ?g of total RNA was used for cDNA syntheses. Arabidopsis thaliana mRNAs (Stratagene, La Jolla, CA) were introduced as internal probe standards into reverse transcription reactions to normalize data between different arrays. Cy3- or Cy5-fluorescently labeled cDNAs were hybridized onto the NIA 15K mouse cDNA prefabricated arrays (8), supplied by the Massachusetts Institute of Technology (MIT)-BioMicro Center, using the procedure provided by the MIT-BioMicro Center. Hybridized microarrays were scanned with the arrayWoRxeTM Biochip Reader (Applied Precision LLC, Northwest Issaquah, WA). The fluorescence intensity of each spot was analyzed from the scanned tiff images by using the DigitalGenomeTM software (MolecularWare, Inc. Cambridge, MA). The Cy3 and Cy5 fluorescence intensities were normalized by calculating the normalization factor from total intensity normalization (10). Analyses for each self-renewal pattern comparison were performed as duplicate independent experiments. For each comparison, we performed two chip hybridizations with reciprocally labeled Cy3 or Cy5 target cDNAs to each biological sample. The entire analysis incorporated data from 16 independent chips. A gene was selected for data analyses only if the mean value of foreground pixels of the spot was greater than the sum of the mean and two standard deviations of the background pixels. For individual gene probe spots, the expression intensities of Cy5 and Cy3 channels were estimated by subtracting mean backgrounds from mean foregrounds. The ratios of the final gene expression intensities for the asymmetrically self-renewing states to the respective symmetrically self-renewing states were calculated. These ratio values (7) were used for sparse feature selection. References: 1. M. Noh, J. L. Smith, Y. H. Huh, J. L. Sherley, J. L., A resource for discovering specific and universal biomarkers for distributed stem cells. PLoS ONE 6(7): e22077. doi:10.1371/journal.pone.0022077 (2011). 2. J. L. Sherley, P. B. Stadler, D. R. Johnson, Expression of the wild-type p53 antioncogene induces guanine nucleotide-dependent stem cell division kinetics. Proc. Natl. Acad. Sci. USA 92, 136-140 (1995). 3. Y. Liu, S. A. Bohn, J. L. Sherley, Inosine-5’-monophosphate dehydrogenase is a rate-limiting factor for p53-dependent growth regulation. Mol. Biol. Cell 9, 15-28 (1998). 4. L. Rambhatla et al., Cellular senescence: ex vivo p53-dependent asymmetric cell kinetics. J. Biomed. Biotech. 1, 28-37 (2001). 5. L. Rambhatla, S. Ram-Mohan, J. J. Cheng, J. L. Sherley, Immortal DNA strand co-segregation requires p53/IMPDH-dependent asymmetric self-renewal associated with adult stem cells. Cancer Research 65, 3155-3161 (2005). 6. Y. Liu et al., Comparison of bax, waf1, and IMP dehydrogenase regulation in response to wild-type p53 expression under normal growth conditions. J. Cell Physiol. 177, 364-376 (1998). 7. M. Noh, thesis, Massachusetts Institute of Technology (2006). 8. T. S. Tanaka et al., Genome-wide expression pr
2014-06-30 | E-GEOD-40183 | biostudies-arrayexpress