Journal: Microbial Cell Factories

Article Title: Purification of target proteins from intracellular inclusions mediated by intein cleavable polyhydroxyalkanoate synthase fusions

doi: 10.1186/s12934-017-0799-1

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: SHuffle ® T7 express , fhuA2 lacZ::T7 gene1 [lon] ompT ahpC gal λatt: :pNEB3-r1- cDsbC (Spec R , lacI q ) Δ trxB sulA11 R(mcr - 73::miniTn10 –Tet S )2 [dcm] R(zgb - 210::Tn10 –Tet S ) endA1 Δgor ∆(mcrC - mrr)114::IS10 , New England BioLabs.

Techniques: Plasmid Preparation

Journal: Frontiers in Plant Science

Article Title: Receptor-Like Kinases BAK1 and SOBIR1 Are Required for Necrotizing Activity of a Novel Group of Sclerotinia sclerotiorum Necrosis-Inducing Effectors

doi: 10.3389/fpls.2020.01021

Figure Lengend Snippet: The effect of virus-induced gene silencing (VIGS) of NbSOBIR1 and NbBAK1 on the necrotizing activity of Sclerotinia sclerotiorum necrosis-inducing effectors. (A) Nicotiana benthamiana SOBIR1 and BAK1 expression after VIGS treatment as determined by qRT-PCR analysis. Expression is relative to actin as an endogenous control. Means and standard errors of three biological replicates are shown. (B) N. benthamiana plants were subjected to VIGS using the TRV-based vectors ( TRV : NbBAK1 and TRV : NbSOBIR1 ) and leaves infiltrated with Agrobacterium tumefaciens strains carrying effector constructs three weeks after TRV infiltration. TRV : GFP was used as a VIGS negative control. Bcl2-Associated protein X (BAX) was used as a BAK1/SOBIR1-independent positive control (P) for necrosis induction and A. tumefaciens carrying an empty vector as a negative control (N). Leaves in each row were infiltrated with the four constructs at the same locations as shown for the first leaf. Arrows show lesions that were smaller than the control plants. (C) The effect of NbSOBIR1 and NbBAK1 silencing on the necrotizing activity of recombinant SsNE2. N. benthamiana plants were subjected to VIGS using TRV : NbBAK1 and TRV : NbSOBIR1 . TRV : GFP was used as a VIGS negative control. Leaves were infiltrated with recombinant SsNE2 expressed in Escherichia coli three weeks after TRV infiltration. Bcl2-Associated protein X (BAX) was used as a BAK1/SOBIR1-independent positive control (P) for necrosis induction and A. tumefaciens carrying an empty vector as a negative control (N).

Article Snippet: The construct was verified by sequencing and transformed into E. coli SHuffle ® T7 (New England Biolabs, Massachusetts, USA).

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Construct, Negative Control, Positive Control, Plasmid Preparation, Recombinant

Journal: eLife

Article Title: Altered expression of a quality control protease in E. coli reshapes the in vivo mutational landscape of a model enzyme

doi: 10.7554/eLife.53476

Figure Lengend Snippet: DHFR cellular abundance is calculated from lysate activity (see Materials and methods). ER2566 is the parental strain (–Lon). SMT102, SMT201, SMT202, SMT205 denote plasmid constructs with altered promoters and ribosome binding sites (see ) in the ER2566 ∆folA/∆thyA strain. DHFR abundances in ER2566 and ER2566 ∆folA/∆thyA –Lon lysates are colored in grey. DHFR abundances in ER2566 ∆folA/∆thyA +Lon lysates are colored in green. Error bars represent the cumulative percent error (standard deviation) from three independent experiments for velocity and three biological replicates for lysate activity.

Article Snippet: Strain, strain background ( Escherichia coli ) , ER2566 , New England Biolabs , Cat# C2566I , Chemically competent cells.

Techniques: Activity Assay, Plasmid Preparation, Construct, Binding Assay, Standard Deviation

Journal: eLife

Article Title: Altered expression of a quality control protease in E. coli reshapes the in vivo mutational landscape of a model enzyme

doi: 10.7554/eLife.53476

Figure Lengend Snippet: ( A ) Turbidostat schematic. Reoccurring dilutions with fresh medium keep the culture optical density (OD600) below 0.075. ( B ) The selection coefficient for each mutant is the slope of the linear regression of allele frequency over time. The wild-type (squares) value is normalized to zero. Advantageous (red) mutations increase and disadvantageous (blue) mutations decrease in frequency. ( C ) Selection coefficients from deep mutational scanning as a function of enzymatic velocity for purified DHFR point mutants measured in vitro. Velocities at 20 µM DHF were calculated from Michalis-Menten parameters. Error bars reflect the standard deviation from three biological replicates. ( D ) Histogram of selection coefficients. The wild-type value is indicated with a vertical black line. The median standard deviation over all mutations is the cut-off for WT-like behavior (Materials and methods, , ) and is indicated with dashed lines. Mutation are colored as advantageous (red), disadvantageous (blue), WT-like (white), or null (grey). ( E ) Structural model of DHFR (PDB ID: 3QL3) with cross-section slices ( a–e ) indicated. The DHF substrate (green) and the NADPH cofactor (purple) are represented by spheres (yellow carbons and heteroatom coloring). An arrow indicates the perspective for each slice. ( a–e ) five cross-section slices. Color scale indicates numbers of advantageous mutations at each position. Crosshatching indicates residues with >20% solvent accessible surface area. Figure 1—source data 1. Soluble DHFR expression levels in molecules per cell measured from lysate activity assays as described in Materials and methods. The location of the DHFR gene is listed in parenthesis in the first column. Expression values corresponds to the cell strain in the column heading. Figure 1—source data 2. Selection coefficients for –Lon selection compared to monoculture growth rates measured in a plate reader in ER2566 ∆folA/∆thyA (–Lon) as described in Materials and methods. For values listed as ND, no detectable change in OD was measured during a 30 hr growth period. Figure 1—source data 3. Michaelis-Menten kinetics for the set of DHFR mutants ( ; ; ) used to calibrate the selection are reported together with the reference from which the values were taken.

Article Snippet: Strain, strain background ( Escherichia coli ) , ER2566 , New England Biolabs , Cat# C2566I , Chemically competent cells.

Techniques: Selection, Mutagenesis, Purification, In Vitro, Standard Deviation, Expressing, Activity Assay

Journal: eLife

Article Title: Altered expression of a quality control protease in E. coli reshapes the in vivo mutational landscape of a model enzyme

doi: 10.7554/eLife.53476

Figure Lengend Snippet: ( A ) Lysate activity for DHFR variants under selection growth conditions (see Materials and methods) plotted as the rate of change in DHF concentration as a function of time monitored over the window of DHF concentration from 30 µM to 20 µM. DHFR activities in ER2566 ∆folA/∆thyA –Lon lysates are colored in grey. DHFR activities in ER2566 ∆folA/∆thyA +Lon lysates are colored in green. Error bars represent ±1 standard deviation from three biological replicates. ( B ) Relative lysate activities for DHFR variants. Lysate activities from A) normalized by WT-level of activity in the corresponding ±Lon cell lysate.

Article Snippet: Strain, strain background ( Escherichia coli ) , ER2566 , New England Biolabs , Cat# C2566I , Chemically competent cells.

Techniques: Activity Assay, Selection, Concentration Assay, Standard Deviation

Journal: eLife

Article Title: Altered expression of a quality control protease in E. coli reshapes the in vivo mutational landscape of a model enzyme

doi: 10.7554/eLife.53476

Figure Lengend Snippet: Relative expression of DHFR variants. DHFR abundances from normalized by WT-level of abundance in the corresponding ±Lon cell lysate. Relative DHFR abundances in ER2566 ∆folA/∆thyA –Lon lysates are colored in grey. Relative DHFR abundances in ER2566 ∆folA/∆thyA +Lon lysates are colored in green. Error bars represent the cumulative percent error (standard deviation) from three independent experiments for velocity and three biological replicates for lysate activity.

Article Snippet: Strain, strain background ( Escherichia coli ) , ER2566 , New England Biolabs , Cat# C2566I , Chemically competent cells.

Techniques: Expressing, Standard Deviation, Activity Assay

Journal: eLife

Article Title: Altered expression of a quality control protease in E. coli reshapes the in vivo mutational landscape of a model enzyme

doi: 10.7554/eLife.53476

Figure Lengend Snippet: Correlation between the ratio of cellular DHFR abundance ( , , [DHFR] +Lon /[DHFR] –Lon ) and in vivo ∆selection coefficients ±Lon for DHFR wild-type and point mutants. Points are colored by the mutation’s location within the hot spots from and . X-axis error bars represent the cumulative percent error (standard deviation) from three measurements of DHFR concentration with and without Lon (Materials and methods). Y-axis error bars the cumulative error (standard deviation) from three biological replicates for selection with and without Lon (Materials and methods). The ratio of expression for WT is not 1.0 because there is an increase in WT DHFR expression in ER2566 ∆folA/∆thy +Lon relative to WT expression in ER2566 ∆folA/∆thy –Lon (see , ). The reason for the unusual behavior of L24V (positive ∆selection coefficient), a mutation in the active site, is unknown.

Article Snippet: Strain, strain background ( Escherichia coli ) , ER2566 , New England Biolabs , Cat# C2566I , Chemically competent cells.

Techniques: In Vivo, Standard Deviation, Concentration Assay, Selection, Expressing, Mutagenesis

Journal: eLife

Article Title: Altered expression of a quality control protease in E. coli reshapes the in vivo mutational landscape of a model enzyme

doi: 10.7554/eLife.53476

Figure Lengend Snippet:

Article Snippet: Strain, strain background ( Escherichia coli ) , ER2566 , New England Biolabs , Cat# C2566I , Chemically competent cells.

Techniques: Recombinant, Plasmid Preparation, Expressing, In Vivo, Concentration Assay, Western Blot, Selection, Sequencing, Amplification