bbsi digested vector  (New England Biolabs)

Journal: bioRxiv

Article Title: Slow scission of single synaptic vesicles by Dynamin at physiological temperature

doi: 10.1101/2025.03.18.643914

Figure Lengend Snippet: a, Normalized average fluorescent traces of Dynamin I-EGFP overexpressed in Xenapses responding to a 50 AP 40 Hz stimulus at RT (black) and 37°C (red). mean ± s.e.m. of n = 19 measurements at RT vs n = 27 measurements at 37°C both from 5 biological replicates. b, Normalized average trace of single Dynamin events elicited with 1 or 2 AP. mean ± s.e.m. c, Normalized cumulative event duration distribution (pORANGE Dynamin I-EGFP knock-in) for RT (red) and 37°C (black). For 37°C, 1031 events (evoked with 1 or 2 AP) consisting of n = 16 measurements from 9 biological replicates. For RT, 119 events (evoked with 2 or 5 AP) consisting of n = 10 measurements from 2 biological replicates.

Article Snippet: These were then individually ligated into BbsI digested pORANGE cloning template vector (Addgene Plasmid #131471) enabled by the duplex having CACCG and AAAC overhangs at either end.

Techniques: Knock-In

Journal: bioRxiv

Article Title: Slow scission of single synaptic vesicles by Dynamin at physiological temperature

doi: 10.1101/2025.03.18.643914

Figure Lengend Snippet: a, Schematic depicting EGFP targetting to the Dynamin I gene by CRISPR-Cas9 based pORANGE strategy (BSE : Bundle signalling element, PRD : Proline rich domain). b, TIRF-microscopy image 3 s after 1 AP stimulus of Xenapses expressing EGFP tagged to endogenous Dynamin I with the Dynamin puncta circled in red; scale bar, 3 µm. c, Representative traces of individual evoked Dynamin recruitment and scission events occurring at various time points after the stimulus. d, An exemplar trace showing the stepwise Dynamin recruitment and annotated with measurable properties. Fit (red) obtained by applying the Autostepfinder step-detection function. Shown below are fluorescence images of the single Dynamin scission event at times indicated; scale bar, 0.5 µm. e, Event duration distribution, f, Event latency distribution and g , Event time to scission distribution. e,f,g are from the same data set of 1129 events in response to 1 or 2 AP and analyzed from n = 16 measurements from 7 biological replicates.

Article Snippet: These were then individually ligated into BbsI digested pORANGE cloning template vector (Addgene Plasmid #131471) enabled by the duplex having CACCG and AAAC overhangs at either end.

Techniques: CRISPR, Microscopy, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Slow scission of single synaptic vesicles by Dynamin at physiological temperature

doi: 10.1101/2025.03.18.643914

Figure Lengend Snippet: Representative traces of pre-stimulus spontaneous (a) and straddle (b) Dynamin recruitment and scission events. c, d, Average trace of spontaneous single Dynamin events centered in space and time along the scission frame and the corresponding average trace of Clathrin fluorescence (d) . Inset images show normalized average fluorescence at time points indicated. mean ± s.e.m. of 38 events from n = 7 measurements with both sets of data from 3 biological replicates. e, Normalized cumulative event duration distributions for Dynamin I EGFP knock-in by pORANGE for evoked (black) and spontaneous events (red, including straddle events). 1031 evoked and 190 spontaneous events both from same data set consisting of n = 16 measurements and 9 biological replicates. f, Normalized inverse cumulative distribution of scission waiting times (time course of endocytosis) for all Dynamin scission events (solid red) and excluding straddle events (dashed red). A monoexponential fit (black) to the decaying phase after 4 seconds (median duration time) yields a time constant of 9.64 seconds. 206 evoked (1 AP) events and 27 straddle events both from n = 4 measurements and biological replicates.

Article Snippet: These were then individually ligated into BbsI digested pORANGE cloning template vector (Addgene Plasmid #131471) enabled by the duplex having CACCG and AAAC overhangs at either end.

Techniques: Fluorescence, Knock-In

Journal: bioRxiv

Article Title: Slow scission of single synaptic vesicles by Dynamin at physiological temperature

doi: 10.1101/2025.03.18.643914

Figure Lengend Snippet: a, Schematic depicting EGFP insertion to the Dynamin I 3‘UTR by CRISPR-Cas9 based TKIT strategy. b, Normalized cumulative stimulus-evoked event duration for Dynamin I EGFP-tagged by pORANGE (black) and TKIT(red) knock-in strategies. 118 events from n = 6 measurements and 2 biological replicates with post-stimulus events evoked at 2 or 5 AP. c, Representative TIRFM image of a Xenapse formed on a 2.5 µm micropattern expressing pORANGE tagged Dynamin-EGFP. Several puncta of Dynamin recruitment are visible 1 s after 2 AP; scale bar, 1 µm. d, Normalized cumulative event durations including straddle events for Xenapses growing on 5 µm (black) and 2.5 µm (red) micropatterns. e , Normalized inverse cumulative distribution of waiting times of scission (time course of endocytosis) for Xenapses growing on 5 µm (black) and 2.5 µm (red) micropatterns. For pORANGE (b) and 5 µm spots (d,e) , same data set as for post-stimulus in Fig.3f . and for 2.5 µm spots (d,e) , 65 events from n = 7 measurements and 2 biological replicates with post-stimulus events evoked at 1, 2 or 5 AP.

Article Snippet: These were then individually ligated into BbsI digested pORANGE cloning template vector (Addgene Plasmid #131471) enabled by the duplex having CACCG and AAAC overhangs at either end.

Techniques: CRISPR, Knock-In, Expressing

Journal: bioRxiv

Article Title: Slow scission of single synaptic vesicles by Dynamin at physiological temperature

doi: 10.1101/2025.03.18.643914

Figure Lengend Snippet: a, Event latency distribution for Dynamin I-EGFP (pORANGE knock-in) scission events in response to 1 (n = 206), 2 (n = 208) and 5 AP (n = 233). n = 4 measurement from 3 biological replicates for 1 AP and n = 6 measurements from 5 biological replicates for 2 and 5 AP. b , SEM images of the two major observed classes of Clathrin coated structures, domed and pit, and the transition between the two. c , Density of domed (8.03 ± 3.62 per mm 2 ) and pit (0.81 ± 0.36 per mm 2 ) structures from SEM images of unroofed Xenapses (n = 17).

Article Snippet: These were then individually ligated into BbsI digested pORANGE cloning template vector (Addgene Plasmid #131471) enabled by the duplex having CACCG and AAAC overhangs at either end.

Techniques: Knock-In