t reesei tu 6  (ATCC)

Journal: Biotechnology for Biofuels

Article Title: A GFP-fusion coupling FACS platform for advancing the metabolic engineering of filamentous fungi

doi: 10.1186/s13068-018-1223-8

Figure Lengend Snippet: Schematic illustrating the GFP-fusion coupling FACS platform accelerating the heterologous gene expression process in T. reesei. a gfp gene was fused to the target gene and acted as the indicator for successful expression of heterologous genes and for spore separation through an FACS platform. Heterologous genes of unsuccessful expressions would be excluded following flow cytometry analysis, since no fluorescence was detected. For genes with successful expression, strains with gene expressed at expected levels can be rapidly obtained from a large candidate pool through cell sorting, as well as an additional confirmation of hyphae fluorescence on 96-well plate cultures. Time and numbers marked in the figure indicated the time and numbers of selected transformants in each procedure. b Cellular fatty acyl-CoA could be converted to fatty alcohol by functional fatty acyl-CoA reductase (FAR). The FAR viability was monitored using FAR fused with GFP with FAR expression levels being reflected by GFP fluorescence intensity

Article Snippet: T. reesei strain QM9414 (ATCC 26921) and a uridine auxotrophic T. reesei strain TU-6 (ATCC MYA256) were used in this study.

Techniques: Gene Expression, Expressing, Flow Cytometry, Fluorescence, FACS, Functional Assay

Journal: Biotechnology for Biofuels

Article Title: A GFP-fusion coupling FACS platform for advancing the metabolic engineering of filamentous fungi

doi: 10.1186/s13068-018-1223-8

Figure Lengend Snippet: Flow cytometry analysis of T. reesei cells harboring gfp -fused gene construct. Trpdi2 was selected as the homologous gene, which was confirmed as expressing functionally, for feasibility of flow cytometry analysis of T. reesei cells with gene– gfp -fusion construct. Both spores ( a , b ) and protoplast ( c , d , generated from hyphae by enzymatic degradation) were utilized for the evaluations. pyr4 -TU-6 strain served as the negative control in analyzing the fluorescence distribution of Trpdi2 - gfp -TU-6 strain’s cell population. 100,000 or 30,000 cells were analyzed for spore or protoplast samples, respectively. Dashed and light-blue lines marking the same value of GFP-Log_Height were for direct comparisons of results from different panels. FSC forward scatter

Article Snippet: T. reesei strain QM9414 (ATCC 26921) and a uridine auxotrophic T. reesei strain TU-6 (ATCC MYA256) were used in this study.

Techniques: Flow Cytometry, Construct, Expressing, Generated, Negative Control, Fluorescence

Journal: Biotechnology for Biofuels

Article Title: A GFP-fusion coupling FACS platform for advancing the metabolic engineering of filamentous fungi

doi: 10.1186/s13068-018-1223-8

Figure Lengend Snippet: Flow cytometry analysis of T. reesei spores constitutively expressing gfp. GFP was driven by three strong promoters on originally constructed plasmids designed for convenient cloning (multiple cloning site) and multiple segment assembly (BioBrick assembly). These plasmids were co-transformed into TU-6 with P19- pyr4 for transformant’s spore collection. 100,000 cells were analyzed for each sample. Two batches of analysis were performed, with the same indications of the variation between samples. Dashed and light-blue lines marking the same value of GFP-Log_Height were for direct comparisons of results from different panels. FSC forward scatter

Article Snippet: T. reesei strain QM9414 (ATCC 26921) and a uridine auxotrophic T. reesei strain TU-6 (ATCC MYA256) were used in this study.

Techniques: Flow Cytometry, Expressing, Construct, Cloning, Transformation Assay

Journal: Biotechnology for Biofuels

Article Title: A GFP-fusion coupling FACS platform for advancing the metabolic engineering of filamentous fungi

doi: 10.1186/s13068-018-1223-8

Figure Lengend Snippet: Flow cytometry analysis of functional viability of fatty acyl-CoA reductases (FARs) in T. reesei. Spores harboring heterologous genes encoding FARs driven by the pdc promoter were analyzed for the evaluation of functional viable FARs in T. reesei . pyr4 -TU-6 and Ppdc- gfp -TU-6 strains were used as the negative and positive controls, respectively. Gates marking the same value range were defined according to the fluorescence distribution of positive cells of Ppdc- gfp -TU-6 spores. Numbers indicate the mean ratio of gate-defined spore number to that of the whole-cell population, and standard deviation was calculated from triplicate analysis on transformant spore pools. 100,000 cells were analyzed for each sample. Two batches of experiments were performed, and significant differences were only observed between pyr4 -TU-6 and Ppdc- Tafar1 - gfp -TU-6 for FAR-expressing spores. FSC forward scatter

Article Snippet: T. reesei strain QM9414 (ATCC 26921) and a uridine auxotrophic T. reesei strain TU-6 (ATCC MYA256) were used in this study.

Techniques: Flow Cytometry, Functional Assay, Fluorescence, Standard Deviation, Expressing

Journal: Biotechnology for Biofuels

Article Title: A GFP-fusion coupling FACS platform for advancing the metabolic engineering of filamentous fungi

doi: 10.1186/s13068-018-1223-8

Figure Lengend Snippet: Fatty alcohol production profiles of engineered T. reesei strain in shake-flask fermentation. Spores of the engineered strain were inoculated and cultured on medium without ( a ) or with ( b ) dodecane addition for cultivations and detections. Intracellular and extracellular fatty alcohol (FAL, hexadecanol and octadecanol), glucose, and biomass were detected over 144 h. Results are the mean of three replicates and error bars indicate standard deviations ( n = 3 ± SD)

Article Snippet: T. reesei strain QM9414 (ATCC 26921) and a uridine auxotrophic T. reesei strain TU-6 (ATCC MYA256) were used in this study.

Techniques: Cell Culture

Journal: Biotechnology for Biofuels

Article Title: A GFP-fusion coupling FACS platform for advancing the metabolic engineering of filamentous fungi

doi: 10.1186/s13068-018-1223-8

Figure Lengend Snippet: Correlation of TaFAR1-fused GFP intensity and fatty alcohol production in T. reesei. Flow cytometry-sorted spores were cultivated and subjected to fatty alcohol quantification ( a ) and hyphae fluorescence detection ( b ). Strains were grouped according to the fluorescence value/OD600 ( b ). 10 or 1 strain from each group was randomly selected for fatty alcohol quantification ( a , b ) or Tafar1 expression detection ( c ), respectively. Results of hexadecanol yield are the mean of experiments on ten separate strains and error bars indicate standard deviations ( n = 10 ± SD). Asterisks indicated a significant difference ( p < 0.05) according to Student’s t test. Con: pyr4 -TU-6 strain, G6: Ppdc- gfp -TU-6 strain, T1, T2, T3: Ppdc- Tafar1 - gfp -TU-6 strains randomly selected from groups with different fluorescence value/OD600 (200–300, 300–400, 400–450), respectively

Article Snippet: T. reesei strain QM9414 (ATCC 26921) and a uridine auxotrophic T. reesei strain TU-6 (ATCC MYA256) were used in this study.

Techniques: Flow Cytometry, Fluorescence, Expressing