Journal: Molecular Systems Biology

Article Title: Identification of novel toxins associated with the extracellular contractile injection system using machine learning

doi: 10.1038/s44320-024-00053-6

Figure Lengend Snippet: ( A ) A representative drop assay of the EATs that were cytotoxic to E. coli . Ctr - bacteria with empty pBAD24 plasmids. Glucose leads to cloned gene repression and arabinose leads to its induction. Bacteria were serially diluted from left to right to quantify CFUs. ( B ) A representative drop assay of yeast EATs. Ctr - yeast cell with empty pESC plasmids. Glucose leads to cloned gene repression and galactose leads to its induction. Yeasts were serially diluted from left to right to quantify CFUs. ( C ) Schematic representation and annotation of each validated predicted EAT and its cognate eCIS operon. .

Article Snippet: Candidate EAT gene sequences were retrieved from IMG, synthesized with codon optimized for expression in E. coli , and cloned into pBAD24 (a gift from Yechiel Shai, Weizmann Institute of Science) by Twist Bioscience (South San Francisco).

Techniques: Bacteria, Clone Assay

Journal: PLoS ONE

Article Title: Members of the Rid protein family have broad imine deaminase activity and can accelerate the Pseudomonas aeruginosa D-arginine dehydrogenase (DauA) reaction in vitro

doi: 10.1371/journal.pone.0185544

Figure Lengend Snippet: Strains and plasmids.

Article Snippet: Primers used to generate plasmids are listed in . pDM1474 (pBAD24-ACIAD3089) was synthesized by GenScript (Piscataway, NJ) and the coding sequence was codon optimized for expression in Escherichia coli .

Techniques: Plasmid Preparation, Expressing, Over Expression

Journal: PLoS ONE

Article Title: Members of the Rid protein family have broad imine deaminase activity and can accelerate the Pseudomonas aeruginosa D-arginine dehydrogenase (DauA) reaction in vitro

doi: 10.1371/journal.pone.0185544

Figure Lengend Snippet: Primers used in plasmid construction.

Article Snippet: Primers used to generate plasmids are listed in . pDM1474 (pBAD24-ACIAD3089) was synthesized by GenScript (Piscataway, NJ) and the coding sequence was codon optimized for expression in Escherichia coli .

Techniques: Plasmid Preparation, Sequencing

Journal: Cell

Article Title: Structure of the Type VI secretion system contractile sheath

doi: 10.1016/j.cell.2015.01.037

Figure Lengend Snippet: (A) Sheath assembly was detected by fluorescence microscopy. Parental strain - V. cholerae with VipA-msfGFP fusion encoded in the native locus. Deletion of vipB gene was complemented by expression of either wt vipB or vipB lacking C-terminal β-strand (VipB-ΔC) from pBAD24 plasmid. 15×10 µm fields of cells are shown. Bar is 1 µm. See also Supplemental Movie S2. (B) Expression of VipB or VipB-ΔC was detected in the indicated strains prepared as for the imaging shown in (A) by western-blotting using VipB specific antibody. (C) Level of E. coli killing on a plate was measured for indicated strains after 3 h of incubation at 10:1 ratio. Presence or absence of vipA or vipB on the chromosome is indicated by “−” or “+”, respectively. Complementation was from pBAD24 plasmid carrying indicated genes. ΔN – vipA lacking N-terminal β-strand; ΔC – vipB lacking C-terminal β-strand. Data represented as mean +/− standard deviation. (D) Sheath assembly was detected by fluorescence microscopy. Parental strain - V. cholerae vipA−. Deletion of vipA gene was complemented by expression of either wt vipA or vipA lacking N-terminal β-strand (VipA-ΔN) from pBAD24 plasmid. 20×20 µm field of cells shown. Bar is 1 µm. See also Supplemental Movie S3. (E) Dynamics of sheath assembly for wild type VipA (2 examples, top) and VipA lacking N-terminal β-strand (VipA-ΔN) (2 examples, middle). An example of sheath contraction and disassembly shown for VipA-ΔN (bottom).

Article Snippet: The linker between VipA and msfGFP was 3xAla 3xGly as used previously on pBAD24 plasmid ( Basler et al., 2012 ).

Techniques: Fluorescence, Microscopy, Expressing, Plasmid Preparation, Imaging, Western Blot, Incubation, Standard Deviation