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Deep Representation Learning Improves Prediction of LacI-mediated Transcriptional Repression

ABSTRACT: We analyze LacI mutations with the goal to understand and better predict basal transcriptional repression function after mutation. This study utilized lac repressor variants from the Taylor, et al. 2015 Nature Methods study including ten tiles of synthesized single mutations (NCBI GEO Series GSE75009), and an additional six tiles corresponding to higher-order mutations (this study).

ORGANISM(S): Escherichia coli

PROVIDER: GSE175456 | GEO | 2021/06/22

REPOSITORIES: GEO

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Project description:Salmonella is an important enteric pathogen that causes a spectrum of diseases varying from mild gastroenteritis to life threatening typhoid fever. Salmonella does not have lac operon. However, E. Coli, Salmonellaâs close relative, has lac operon. Being an enteric pathogen like E. coli, Salmonella will also benefit from lac operon. Then, why Salmonella has lost lac operon?. To address this question, lacI, an important component of lac operon was expressed in Salmonella via pTrc99A plasmid. As a control, pTrc99A without lacI was also expressed in Salmonella. The effect of LacI on the transcription profile of Salmonella was analyzed using microarray technique. Overall design The total RNA was isolated from Salmonella using âRNeasy Mini Kitâ (QIAGEN). Organism used: Salmonella enterica serovar Typhimurium LT2 * Slides: Agilentâs arrays (8x15k) AMADID: NO: 17809 * Starting material: Salmonella RNA in nuclease-free water * RNA Samples used: Wild type (WT), WT + pTrc99A+LacI (PTRC), WT + pTrc99A-LacI(Delta lac) * Labeling kit: MessageAmpâ¢ II-Bacteria RNA Amplification Kit from Ambion Cat # AM1790 * Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA * Total RNA and cRNA Purification Kit: Qiagenâs RNeasy minikit Cat#74104 * Hybridization Kit: Agilentâs In situ Hybridzation kit 5184-3568 * RNA quality was checked using Bioanalyzer. The array slides were scanned immediately by PerkinElmer Scan array Gx Microarray scanner. The Scan array software (PerkinElmer) was used for grid wise normalization of array images. Two arrays were used with mutant and rescue experiments and dye swap experiments were included in the final analysis. The data was analysed by GeneSpring GX and Biointerpreter software from Genotypic Technology, Bangalore. The differential expression was considered if the Log 2 mean of at least -1 for the down regulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from both replicates.

Project description:Clostridium thermocellum is a candidate for cellulosic ethanol production. It expresses enzymes for both cellulose solubilization and its fermentation to produce lignocellulosic ethanol. Understanding how this organism regulates gene expression is of importance for developing a better fundamental understanding of this industrial relevant bacterium. We are primarily interested in gene regulation by three predicted LacI regulators. A previous report of one LacI regulator in C. thermocellum ATCC27405 (Cthe_2808) indicated this was a transcriptional repressor of neighboring genes with repressor activity relieved in the presence of laminaribiose (a β-1,3 disaccharide). The current work is aimed at understanding if a homolog of this LacI transcriptional regulator in C. thermocellum DSM1313 and two others putatively characterized as LacI regulators are in fact global regulatory proteins with extensive regulons or, if like the majority of LacI transcription factors, the regulation is limited to local genomic regions. Each of the three LacI regulators was deleted in C. thermocellum DSM1313. Genome re-sequencing was used to confirm the deletion and ensure nonspecific recombination events were avoided during the gene deletion strategy. Growth of each strain on cellobiose was unaffected by any of the gene deletions under pH controlled fermentations. Global gene expression patterns taken at mid-log phase for each strain identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly up-regulated (up to 9 fold) in the absence of each LacI regulator. Thus suggesting these were repressed under wild type conditions. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters with putative motifs ranging from 16-18 bp. Work is ongoing to confirm the specific binding motif recognized by the two previously un-characterized transcription factors and assess the occurrence of this motif in the C. thermocellum DSM1313 genome. The identification of LacI repressor activity on hemicellulose gene expression is a key result of this work and will add to the small body of existing literature on the area of gene regulation in C. thermocellum.

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Deep Representation Learning Improves Prediction of LacI-mediated Transcriptional Repression

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