Project description:Populations of engineered metabolite-producing microorganisms are prone to evolutionary production declines during industrial-scale cultivations. In this study, we develop a synthetic product addiction system in E coli that addicts mevalonic acid production cells to mevalonic acid. Through experimentally simuluated long-term fermentation, we investigate how product-addicted organisms remain stable and avoid formation of genetic subpopulations of fit, non-producing cells.
Project description:Understand the mechanisms of evolution in large-scale bio-production by tracking population dynamics leading to production decline in mevalonic acid-producing Escherichia coli. Industrial bioproduction entails growth of the production host to large bioreactors (e.g. 1-300 m3). This may put the organism at risk for generating non-producing subpopulations of genetic heterogeneity, which is not phenotypically detected at lab-scale (e.g. 2 L). To study these dynamics, we experimentally simulated these growth durations by passing mevalonic acid-producing E. coli to maintain the populations in exponential growth for 45 generations.
Project description:To gain deeper insights into antibacterial mechanisms of NAD+ and bacterial adaptation, we generated and sequenced NAD+ resistant clones of Spn. For this purpose, Spn was cultivated in liquid medium with increasing concentrations (50 µM to 5 mM) of NAD+. After six passages, bacteria were plated on blood agar supplemented with 500 µM NAD+ and three clones were picked
Project description:Caco-2 and HT-29 cells were barcoded using the CloneTracker lentiviral barcode library and then irinotecan and capecitabine resistant derivatives of these cell lines were established. Four million barcoded Caco-2 and HT-29 cell were seeded into 15 cm cell culture dishes. When the cells reached confluency, two million cells per dish were seeded into four different 15 cm dishes with 25 mL medium (DMSO Control, Replica A, B, C) and two million cell pellets were stocked as initial cell population.For Caco-2 cell line, mediums in the dishes were changed twice a week with fresh mediums containing IC50 dose (4 months) and subsequently 2x IC50 dose (2 months) of capecitabine, for HT-29 cell line IC50 dose (6 months) of irinotecan. Caco-2 and HT-29 cell lines treated with DMSO were given the same amount of DMSO used in dissolving compounds as fresh medium. Following the end points of six months for each cell line, DNA isolation from harvested cell lines and collected medium of resistant B cell lines were carried out and barcodes were sequenced.
Project description:HT-29 cells were barcoded using the CloneTracker lentiviral barcode library and then dabrafenib resistant derivative of these cells were established. Five million barcoded HT-29 cells were seeded into 15 cm cell culture dishes. When the cells reached confluency, two million cells per dish were seeded into four different 15 cm dishes (DMSO Control, Replica A, B, C) and two million cell pellets were stocked as initial cell population. Harvesting used medium through the experiment was performed at monthly intervals. Barcoded HT-29 cell line replicates A, B, and C were treated with 2XIC50 (199.6 nM) of dabrafenib concentration for the duration of 3 months. Following the end points of 3 months for each cell line, DNA isolation from harvested cell lines and collected medium of resistant A cell lines were carried out and barcodes were sequenced.
Project description:Aspergillus flavus is a common saprophyte and opportunistic pathogen producing aflatoxin (AF) and many other secondary metabolites. 5-Azacytidine (5-AC), a derivative of nucleoside cytidine, is widely used for studies in epigenetics and cancer biology as an inactivator of DNA methyltransferase and is also used for studying secondary metabolism in fungi. Our previous studies showed that 5-AC affects development and inhibits AF production in A. flavus, and that A. flavus lacks DNA methylation. How this common DNA methyltransferase inhibitor affects development and AF production is not clear. In this study, we applied an RNA-Seq approach to elucidate the mechanism of 5-ACM-bM-^@M-^Ys effect on A. flavus. In our current study, we identified 240 significantly differently expressed (Q-value<0.05) genes after 5-AC treatment, including two backbone genes in secondary metabolite clusters #27 and #35, which are involved in development or survival of sclerotia. With 5-AC treatment, about three quarters of the genes in the AF biosynthetic gene cluster in A. flavus were down-regulated to a certain degree. Strikingly, at least two genes aflI and aflLa, were completely inhibited. Interestingly, several genes involved in fungal development were down-regulated, especially veA, which is a gene that encodes protein bridges VelB and LaeA. This result supports the hypothesis that 5-AC affects development and AF production through weakening or even interrupting the connection between VelB and LaeA and then causing dysregulation of the expression pattern of genes involved in development and secondary metabolism. Our results improved the A. flavus genome annotation, provided a comprehensive view of the transcriptome of A. flavus responding to 5-AC and confirmed that fungal development and secondary metabolism are co-regulated. In additon, the RNA-Seq data of another sample treated with gallic acid was used to improve A. flavus genome annotation. mRNA of Aspergillus flavus cultured in three different culture media PDB, PDB+5-AC(5-Azacytidine),and PDB+GA(gallic acid) was subjected to sequence independently.
Project description:S. meliloti strains with a bi- and monopartite genome configuration were constructed by consecutive Cre/lox-mediated site-specific fusions of the secondary replicons. Beside the correct genomic arrangements, these strains and precursors were tested for variations in the nucleotide sequence. Futher, a marker fequency analysis was performed to test if replication is initiated at all origins and to determine the replication termination regions of the triple replicon fusion molecule. To gain the sequence data for these analyses, respective strains were applied to whole genome sequencing using an Illumina MiSeq-System and Oxford Nanopore (MinION) sequencing technology.
Project description:Recent work has shown that small non-coding RNAs, including miRNAs, serve an important role in controlling gene expression during development and disease. However, little detailed information exists concerning the relative expression patterns of small RNAs during development of C. elegans. Here we use recent advances in high-throughput sequencing technology to show that expression of non-coding small RNAs, including miRNAs, changes dynamically during development and in the different sexes of C. elegans; approximately 16% of known miRNAs changed over 10 fold in expression during C. elegans development and about 12% of miRNAs showed major changes in expression between males and hermaphrodites of C. elegans. These results should lead to a better understanding of the expression and function of small RNAs in C. elegans development. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Examination of small RNA expression in six different developmental stages of hermaphrodites (Embryo, mid-L1, mid-L2, mid-L3, mid-L4, young adult), and young adult males (dpy-28;him-8) and spermatogenesis-defective young adult hermaphrodites (spe-9). The number of sequence reads for miRNA was assessed from the raw sequence data from Solexa sequencing using perfect sequence matching to known miRNAs (miRBase Release 11.0).
Project description:Aflatoxins are carcinogenic fungal secondary metabolites. Levels of aflatoxins in agricultural commodities are stringently regulated by many countries. A cluster of genes is responsible for aflatoxin biosynthesis by Aspergillus flavus and other closely related species. Expression of the clustered aflatoxin genes is governed by a complex network of regulatory mechanisms. To better understand the molecular events that are associated with aflatoxin production, transcription profiling by microarray analyses which compared three independent aflatoxigenic A. flavus strains to individual isogenic progenies that no longer produced aflatoxins after serial transfers was carried out. Twenty-two significantly differentially expressed features were identified. After physical mapping using the A. oryzae genome sequence as the reference, the number of unique genes was reduced to 16. Compared to the parental strains, changes in the aflatoxin gene expression levels in the progenies were not significant, which suggests that the inability to produce aflatoxins is not caused by decreased expression. The only gene showing higher expression levels in the progenies is homologous to glutathione S-transferease genes. Overexpression of this gene, named hcc, at six- to nine-fold in an aflatoxigenic A. flavus did not cause discernible changes in colony morphology or aflatoxin production. Loss of aflatoxin production after serial transfers may not result from a single event but caused by multiple factors. Keywords: Compartiave hybridization toxigenic and atoxigenic lines of Aspergillus Aspergillus flavus NRRL 29459, NRRL 29474, and NRRL 29490 are aflatoxigenic strains originated from soil collection in a peanut field (Terrell Co., Georgia, USA). Strains 459B-20-2, 474A-20, and 499A-20 were nonaflatoxigenic isolates obtained after 20 serial transfers of the parental strains on potato dextrose agar slants (Horn and Dorner 2002). Comparsions in each experiment consisted of one aflatoxigenic parental strain and one nonaflatoxigenic progeny, compared after 48- or 72-hr growth. Each comparison was repeated with duplicate dye-flip.