Journal: mSphere

Article Title: The promiscuous biotin ligase TurboID reveals the proxisome of the T3SS chaperone IpgC in Shigella flexneri

doi: 10.1128/msphere.00553-24

Figure Lengend Snippet: TurboID is a highly effective promiscuous biotin ligase in the cytoplasm of Shigella flexneri . ( A ) Biotinylation was measured at the indicated times after the addition of 50 µM biotin to cultures of S. flexneri strain M90T harboring the empty vector (EV), BioID-Myc (BID), BioID2-Myc (BID2), TurboID-Myc (TID), miniTurbo-Myc (miT). pSU2.1 was used as the backbone vector. The expression of the biotin ligases and the loading controls was measured by immunoblotting using a monoclonal antibody against the Myc tag and a polyclonal antibody against RecA, respectively. The biotinylation blots were performed with the streptavidin-horseradish peroxidase (Strep.). ( B ) Densitometry on three biological replicates ( n = 3) of the biotinylation blot represented in panel A was used to estimate the relative biotinylation activity of the biotin ligases at 10 min after the addition of biotin. The results of a one-way analysis of variance with Tukey’s post hoc test are shown (***, P < 0.001). Error bars represent standard deviation to the mean. ( C ) Biotinylation and Myc blots for a new set of three biological replicates ( n = 3) labeled Rep. 1, 2, or 3, comparing TurboID to miniTurbo 10 min after the addition of biotin. The graphs show the densitometry data for the biotinylation activity (Strep., top panel) and expression (Myc, bottom panel), respectively. The results of Student’s t -tests for unpaired data with equal variance and a 95% CI are shown (***, P < 0.001; ****, P < 0.0001). Error bars represent standard deviation to the mean.

Article Snippet: The coding sequence of bioID , bioID2 , turboID , and miniTurbo [Addgene plasmids #35700 and #74224, kind gift from Kyle Roux ( , ); #107169 and #107170, kind gift from Alice Ting ( )] were amplified by PCR using primers HMIO 607/609, 11/14, 249/251, and 247/251, respectively, which inserted EcoRI (5 ́-end), and XbaI restriction sites and a Myc tag (3 ́-end).

Techniques: Plasmid Preparation, Expressing, Western Blot, Activity Assay, Standard Deviation, Labeling