codon optimized full length ifr1 coding sequence  (GenScript corporation)

Journal: Frontiers in Plant Science

Article Title: Knock-Down of the IFR1 Protein Perturbs the Homeostasis of Reactive Electrophile Species and Boosts Photosynthetic Hydrogen Production in Chlamydomonas reinhardtii

doi: 10.3389/fpls.2017.01347

Figure Lengend Snippet: IFR1 localizes to the cytosol in Chlamydomonas reinhardtii cells. Laser scanning confocal microscopy detection of subcellular localization of the mVenus (yellow) fluorescent reporter fused to N- or C-terminus of IFR1 (N/C). A cell line expressing mVenus in the cytosol (Cyto, ) and the parental strain (PCS) served as controls. Individual imaging channels are presented, YFP: mVenus reporter signal in the yellow range, Chlorophyll: autofluorescence of chlorophyll visualized in the red range and used to orient the cells, Overlay: YFP and Chloro channel overlay, DIC: differential interference contrast. Scale bars represent 5 μm.

Article Snippet: To heterologously express IFR1 in Escherichia coli , a codon optimized full length IFR1 coding sequence (phytozome: Transcript Cre11.g477200.t1.2) was synthesized de novo (Genscript, United States) and cloned between the Nde I and Xho I restriction sites of expression vector pET-24a(+) (Novagen), enabling streptag-based affinity purification.

Techniques: Confocal Microscopy, Expressing, Imaging

Journal: Frontiers in Plant Science

Article Title: Knock-Down of the IFR1 Protein Perturbs the Homeostasis of Reactive Electrophile Species and Boosts Photosynthetic Hydrogen Production in Chlamydomonas reinhardtii

doi: 10.3389/fpls.2017.01347

Figure Lengend Snippet: IFR1 accumulation is triggered by the SOR1-dependent pathway. (A) Samples were taken before (0 h, +S) and during the course of hydrogen production induced by sulfur deprivation of a wild type cell line (16–48 h; –S). IFR1 accumulation was analyzed with an IFR1-specific antiserum (αIFR1) and equal protein loading confirmed by colloidal Coomassie staining (CBB). (B) Comparison of IFR1 mRNA levels in the sor1 mutant and its parental strain (4A+) with samples taken in the late exponential phase. mRNA levels were determined by RTqPCR and the IFR1 transcript level in 4A+ was set to 1. Median and interquartile range shown in the box-and-whisker diagram are derived from two biological replicates, each including nine technical replicates ( n = 18). (C) Representative immunoblot (αIFR1) showing IFR1 accumulation during growth of mutant sor1 and its parental strain (4A+) in nutrient-replete TAP medium for 3 days. Relative band intensities ( Dens. ) determined by densitometric scanning of immunblot signals are given relative to the band intensity of the 4A+ sample at t 48 h (set to 1). (D) Position of the octanucleotide motif CAACGTTG described to represent an electrophile response element (ERE; ) implicated in the genetic response to reactive electrophile species (RES) and SOR1-dependent signaling relative to the start codon (ATG) of the 4.87 kbp IFR1 gene, comprising exons, introns and untranslated regions (UTRs). (E) IFR1 mRNA levels determined by RTqPCR following dark treatment of WT cell cultures with DBMIB (5 μM) and 2-( E )-hexenal (500 μM) for 24 h. The mRNA level of the solvent control sample was set to 1. Median and interquartile range shown in the box-and-whisker diagram are derived from two biological replicates, each including six technical replicates ( n = 12). (F) Immunoblot (αIFR1) showing IFR1 accumulation distinct time points (6–24 h) after the addition of DBMIB (5 μM) or only solvent (Control) to a liquid TAP culture of the C. reinhardtii wild type CC124 and subsequent dark incubation for 24 h.

Article Snippet: To heterologously express IFR1 in Escherichia coli , a codon optimized full length IFR1 coding sequence (phytozome: Transcript Cre11.g477200.t1.2) was synthesized de novo (Genscript, United States) and cloned between the Nde I and Xho I restriction sites of expression vector pET-24a(+) (Novagen), enabling streptag-based affinity purification.

Techniques: Staining, Comparison, Mutagenesis, Whisker Assay, Derivative Assay, Western Blot, Solvent, Control, Incubation

Journal: Frontiers in Plant Science

Article Title: Knock-Down of the IFR1 Protein Perturbs the Homeostasis of Reactive Electrophile Species and Boosts Photosynthetic Hydrogen Production in Chlamydomonas reinhardtii

doi: 10.3389/fpls.2017.01347

Figure Lengend Snippet: IFR1 knock-down causes diminished tolerance toward RES in C. reinhardtii. (A) Immunodetection of IFR1 protein (αIFR1) in the parental strain (PCS; wild type CC124) and IFR1 knock-down strains (IFR1_1 and IFR1_6) detected after 48 h of cultivation in sulfur deplete medium. A colloidal Coomassie stained gel (CBB) served as loading control. Different amounts of proteins were used and band intensities (lower bar diagram) determined by densitometric analysis (1x PCS set to 100%). (B,C) IFR1 accumulation in PCS and IFR1 knock-down strains grown for 24 h in TAP supplemented with DBMIB (5 μM) or 2-(E)-hexenal (500 μM). (D) Growth inhibition by reactive oxygen species determined for the PCS and the two IFR1 knock-down strains during 24 h of growth in TAP supplemented with 4 μM rose Bengal (RB), 15 μM neutral red (NR), or 0.5 μM methyl viologen (MV). Optical densities (determined at 680 and 750 nm) and cell counts are given relative to the untreated/solvent-control sample (set to 1). Error bars indicate standard errors derived from three biological replicates including technical replicates ( n = 3). Asterisks indicate significant differences between PCS and knock-down strains according to a two-tailed Student’s t -test ( p < 0.05). (E,F) Growth inhibition following treatment of PCS and IFR1 knock-down strains with 5 μM DBMIB and 500 μM 2-(E)-hexenal for 9 or 24 h in TAP medium. Standard errors are derived from three biological replicates, including technical replicates ( n = 3). Except for the data indicated by asterisks ( p > 0.05) differences between PCS and knock-down strains were significant according to a two-tailed Student’s t -test ( p < 0.05).

Article Snippet: To heterologously express IFR1 in Escherichia coli , a codon optimized full length IFR1 coding sequence (phytozome: Transcript Cre11.g477200.t1.2) was synthesized de novo (Genscript, United States) and cloned between the Nde I and Xho I restriction sites of expression vector pET-24a(+) (Novagen), enabling streptag-based affinity purification.

Techniques: Knockdown, Immunodetection, Staining, Control, Inhibition, Solvent, Derivative Assay, Two Tailed Test

Journal: Frontiers in Plant Science

Article Title: Knock-Down of the IFR1 Protein Perturbs the Homeostasis of Reactive Electrophile Species and Boosts Photosynthetic Hydrogen Production in Chlamydomonas reinhardtii

doi: 10.3389/fpls.2017.01347

Figure Lengend Snippet: Prolonged hydrogen production in IFR1 knock-down strains compared to the wild type. (A) Time course of hydrogen production for the parental strain (PCS) and IFR1 knock-down strains. Hydrogen yields in the knock-down strains are given relative to the final yield of the parental strain (set to 100%). Each data curve represents an average of three biological replicates including three technical triplicates ( n = 9) with error bars representing the standard error. (B) H 2 production rates during the course of hydrogen production. Error bars indicate the standard error ( n = 9) and asterisks indicate differences between PCS and knock-down strains which are significant according to a two-tailed Student’s t -test ( ∗ p < 0.05).

Article Snippet: To heterologously express IFR1 in Escherichia coli , a codon optimized full length IFR1 coding sequence (phytozome: Transcript Cre11.g477200.t1.2) was synthesized de novo (Genscript, United States) and cloned between the Nde I and Xho I restriction sites of expression vector pET-24a(+) (Novagen), enabling streptag-based affinity purification.

Techniques: Knockdown, Two Tailed Test

Journal: Frontiers in Plant Science

Article Title: Knock-Down of the IFR1 Protein Perturbs the Homeostasis of Reactive Electrophile Species and Boosts Photosynthetic Hydrogen Production in Chlamydomonas reinhardtii

doi: 10.3389/fpls.2017.01347

Figure Lengend Snippet: Contribution of PSII and photosynthetic/respiration (P/R) rates on hydrogen production. (A) Maximum quantum yield (F v /F m ) of dark-adapted cells of the parental strain (PCS) and IFR1 knock-down strains (IFR1_1/IFR1_6) before (t 0 ) and after exposure to sulfur limitation (t 24 –t 168 h ) and aerobic conditions. Error bars indicate the standard error from three biological replicates ( n = 3). (B) Time course of the maximum quantum yield (F v /F m ; left y-axis) and the cellular chlorophyll content (right y-axis) during photosynthetic hydrogen production of the parental strain (PCS) and one of the IFR1 knock-down strains (IFR1_6). Chlorophyll data were normalized to the chlorophyll content of PCS at t 0 (set to 100%). Standard errors derived from three biological replicates ( n = 3) are indicated as error bars. Except for t 0 , the differences between PCS and IFR1_6 in regard to F v /F m were significant according to a two-tailed Student’s t -test ( p < 0.05). (C) Representative immunoblot showing the immunodetection of PSII subunit D1 (upper left panel; αD1) in samples of the parental strain (PCS) and IFR1_6 taken at indicated times during a hydrogen production experiment. A colloidal Coomassie stain (lower left panel; CBB) served as a loading control. Results from densitometric scanning (right panel) of blot signals are given relative to the D1 signal intensity determined for t 0 (set to 100%). Error bars indicate standard errors (three biological replicates; n = 3). (D) Relative H 2 yields obtained with the parental control strain (PCS) (black bars) and knock-down strain IFR1_6 (gray bars) in the absence or presence of 20 μM DCMU. Hydrogen yields determined for the untreated PCS were set to 100%. Error bars represent standard error ( n = 6).

Article Snippet: To heterologously express IFR1 in Escherichia coli , a codon optimized full length IFR1 coding sequence (phytozome: Transcript Cre11.g477200.t1.2) was synthesized de novo (Genscript, United States) and cloned between the Nde I and Xho I restriction sites of expression vector pET-24a(+) (Novagen), enabling streptag-based affinity purification.

Techniques: Knockdown, Derivative Assay, Two Tailed Test, Western Blot, Immunodetection, Staining, Control

Journal: Frontiers in Plant Science

Article Title: Knock-Down of the IFR1 Protein Perturbs the Homeostasis of Reactive Electrophile Species and Boosts Photosynthetic Hydrogen Production in Chlamydomonas reinhardtii

doi: 10.3389/fpls.2017.01347

Figure Lengend Snippet: Knock-down of IFR1 in the boosts hydrogen production in the high hydrogen producer mutant stm6. (A) Immunoblot analysis of IFR1 accumulation in stm6 and stm6_IFR1kd cultivated under sulfur-limiting conditions. Different amounts of total protein (1X; 1.5X; and 2X) were used for immunodetection of IFR1 (αIFR1) with a colloidal Coomassie stain (CBB) serving as a loading control. Results from densitometric signal analysis (dens.) are indicated. (B) Relative time-dependent H 2 yields of the stm6 parental strain (black curve) and stm6_IFR1kd (gray curve) with the final yield in stm6 set to 100%. Error bars represent the standard error (three biological replicates including technical triplicates, n = 9). (C) Maximum quantum yield of PSII determined after dark incubation (Fv/Fm) determined in cultures of stm6 (black bars) and stm6_IFR1kd (gray bars) exposed to sulfur starvation. Standard errors, shown as error bars are derived from three biological replicates including technical duplicates ( n = 6). Except for t 0 , differences between stm6 and stm6_IFR1kd were significant according to a two-tailed Student’s t -test ( p < 0.05).

Article Snippet: To heterologously express IFR1 in Escherichia coli , a codon optimized full length IFR1 coding sequence (phytozome: Transcript Cre11.g477200.t1.2) was synthesized de novo (Genscript, United States) and cloned between the Nde I and Xho I restriction sites of expression vector pET-24a(+) (Novagen), enabling streptag-based affinity purification.

Techniques: Knockdown, Mutagenesis, Western Blot, Immunodetection, Staining, Control, Incubation, Derivative Assay, Two Tailed Test