Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profling of a yeast molecular barcoded ORF library

ABSTRACT: I used Affy Tag4 array as a readout of the molecular barcoded yeast orf library

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Cheuk Hei Ho

PROVIDER: E-MEXP-2077 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Synthetic systems that use positive feedback have been developed to control human disease vectors and crop pests. The tTAV system, which has been deployed in several insect species, relies on a positive feedback circuit that can be inhibited via dietary tetracycline. Although insects carrying tTAV fail to survive until adulthood in the absence of tetracycline, the exact reason for its lethality, as well as the transcriptomic effects of an active positive feedback circuit, remain unknown. Understanding what factors contribute to the variance in tTAV-associated mortality is likely to inform the development of insect control systems. In the present study, the OXI513a tTAV feedback circuit was introduced and verified into D. melanogaster. The tight tetracycline regulation of the system affords a convenient method to conduct a strain-by-strain assessment of the tTAV system and determine the transcriptomic effect, if any, of a positive feedback circuit. Transcriptomic analysis of four independent D. melanogaster tTAV insertion lines, in both adults and larvae, was conducted to examine the transcriptomic influence of the tTAV system. The tTAV lines and non-tTAV control were maintained on TET-on media for 5 generations prior to commencing. Thirty flies, 15 male and 15 female, of the same age, from each of the strains were transferred to either TET-On or TET-Off media. Five days after transfer, adult flies were removed and 10 adult flies, 5 male & 5 female, were frozen at -80°C. Ten larvae from each of these matings were collected at the late second instar stage, the last life stage normally seen prior to lethality, and frozen at -80°C. Three biological replicates were produced for each combination of strain, life-stage, and media. RNA was prepared from frozen samples using Trizol and DNAse was treated using a PureLink RNA Mini Kit (Invitrogen). Microarray experiments employed Agilent Drosophila Gene Expression Microarrays 4x44K and were scanned on an Agilent G2505C scanner (Agilent Technologies). Data collection was divided into two separate experiments. A pilot experiment for strain 102D consisting of two life stages in two conditions each with three biological replicates – a total of 12 samples. The remaining four strains were run as a separate experiment with a total of 48 samples. For each experiment, sample chip position was randomized to avoid genotype- and treatment-specific batch effects.

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Transcription profling of a yeast molecular barcoded ORF library

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