Project description:REPLACE was engineered from an orthogonal alphaviral RNA replication system. It generates a large, continuously diversified library of replicative RNAs through a replicase-limited mode of replication and inducible mutagenesis. We analyzed the mutation frequency at Day0 (IVT RNA), Day1, Day3, Day7, Day14, Day21 and DNA plasmid to estimate the RNA mutation rate. This data was used to guide the construction of RNA mutant libraries.
Project description:REPLACE was engineered from an orthogonal alphaviral RNA replication system. It generates a large, continuously diversified library of replicative RNAs through a replicase-limited mode of replication and inducible mutagenesis. We analyzed the variation of RNA mutations induced by two nucleoside analogues over time and at different concentrations. This data was used to guide the construction of RNA mutant libraries.
Project description:Yeast enolase (Eno2p) conjugated with EGFP and Flag-tag (Eno2p-EGFP-FLAG) and Eno2p with V22A substitution (Eno2V22Ap) conjugated with EGFP and Flag-tag (Eno2V22Ap-EGFP-FLAG) were produced in baker's yeast S. cerevisiae. After semi-anaerobic culture at 30 ˚C for 12h, cells producing Eno2p-EGFP-FLAG formed fluorescent foci, while cells producing Eno2V22Ap-EGFP-FLAG did not. The cells were collected and lysed, and proteins Eno2p-EGFP-FLAG or Eno2V22Ap-EGFP-FLAG and the associated proteins were coimmunoprecipitated using ANTI-FLAG M2 affinity gel and analyzed. Data contain two biological replicates and two technical replicates (n = 4). As the results, 96 proteins were detected with both recombinant Eno2p-EGFP-FLAG and Eno2V22Ap-EGFP-FLAG protein, 29 proteins were detected only with recombinant Eno2p-EGFP-FLAG protein, and 16 proteins were detected only with recombinant Eno2V22Ap-EGFP-FLAG protein. Data Processing/Data Analysis: The separated analytes were detected on an LTQ Velos linear ion trap mass spectrometer (Thermo Scientific). For data-dependent acquisition, the method was set to automatically analyze the five most intense ions observed in the MS scan. The mass spectrometry data were used for protein identification by the Mascot search engine on Protein Discoverer software (ver. 1.2, Thermo Scientific) against the information in the Saccharomyces Genome Database (SGD; http://www.yeastgenome.org). Search parameters for peptide identification included a precursor mass tolerance of 1.2 Da, a fragment mass tolerance of 0.8 Da, a minimum of one tryptic terminus, and a maximum of one internal trypsin cleavage site. Cysteine carbamidomethylation (+57.021 Da) and methionine oxidation (+15.995 Da) were set as a differential amino acid modification. The data were then filtered at a q value ≤ 0.01 corresponding to 1% FDR at the spectral level.
Project description:There are the Mass spectrum original data of Ptc1 and Ptc2. The immunoprecipitated proteins from Ptc1::eGFP and Ptc2::eGFP stains in Aspergillus flavus were detected with SDS-PAGE. Next, in-gel tryptic digestion, and the peptides were obtained and detected by liquid chromatog-raphy coupled with tandem mass spectrometry (LC-MS/MS) on a Q-Exactive (QE) mass spectrometer (Thermo Fisher Scientific)