Mar 06, 2025
Tracking C. elegans behaviour on Agrobacterium
- 1LMS
- Behavioural Genomics
Protocol Citation: e.warren Warren 2025. Tracking C. elegans behaviour on Agrobacterium. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldro9og5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 06, 2025
Last Modified: March 06, 2025
Protocol Integer ID: 123935
Abstract
Tracking of behavioural phenotypes and morphology of C. elegans fed Agrobacterium
Materials
Low peptone NGM:
2% BioAgar, 0.013% Bacto Peptone, 0.3% NaCl, 1mM CaCl2,
1mM MgSO4, 1mM KPO4, 5 µg/mL Cholesterol
Yeast Extract Peptone (YEP) agar:
1% Bacto peptone, 1% Yeast extract 0.5% NaCl, 1.5% BioAgar
Yeast Extract Peptone (YEP) media:
1% Bacto peptone, 1% Yeast extract 0.5% NaCl
Induction Media:
1% NH4Cl, 0.145% MgSO4, 0.15% KCl, 0.01% CaCl2, and 0.0025% FeSO4-7H2O,
2 mM NaPO4 (pH 5.6), 50 mM 2-(4-morpholino)-ethane sulfonic acid (MES), 0.5%
glucose + 100 µM Acetosyringone
LB-Nematode Grown Media (NGM) Agar:
1% tryptone, 0.5% yeast extract, 1% NaCl, 1mM CaCl2, 1mM MgSO4, 1mM KPO4
dx.doi.org/10.17504/protocols.io.bnh2mb8e, 1.7% BioAgar, 5 µg/mL Cholesterol
Plate preparation
Plate preparation
Low peptone (0.013%) NGM https://dx.doi.org/10.17504/protocols.io.2rcgd2w)
was dispensed into each well of a 96-square well plate (Whatman UNIPLATE:
WHAT-77011651) using an Integra VIAFILL https://dx.doi.org/10.17504/protocols.io.bmxbk7in).
The plate was placed in a drying cabinet to lose 3-5% of weight by volume. Wells
were then seeded with 5 μL of E. coli (OP50, OD600 0.1).
Agrobacterium preparation
Agrobacterium preparation
8 days before starting the tracking, Agrobacterium transformed with T-binary vectors containing T-DNA designed to express flp-1 (dna1959), Amyloid-β (dna1977), MmTX1 (dna1948), MmTX1 with signal peptide sequences clec-1 (dna1947), or Spp1(dna1946), controls of wrmScarlet (WS) (dna1401), BFP (dna1621) or no plasmid (Control), was streaked to Yeast Extract Peptone (YEP) agar plates (containing appropriate antibiotics) and grown for 3 days at 28°C.
5 days before starting the tracking, 20 ml YEP media (containing appropriate antibiotics) was inoculated with a single colony of Agrobacterium, and incubated overnight at 28°C and 250 rpm.
4 days before starting the tracking, overnight cultures were diluted 1:100 with YEP media (containing appropriate antibiotics), and cultured at 28°C at 250 rpm until the OD600 reached 0.3.
Agrobacterium was pelleted by centrifugation (5 min 4000g), before resuspending in 1 ml Induction Medium containing 100 µM Acetosyringone.
Samples were shaken at 50 rpm at room temperature for 23 hours.
Coincubation of C. elegans and Agrobacterium
Coincubation of C. elegans and Agrobacterium
4 days before tracking N2 C. elegans were bleach synchronized. dx.doi.org/10.17504/protocols.io.2bzgap6
3 days before starting the tracking, LB-NGM plates (containing appropriate antibiotics) were seeded with 1 ml induced Agrobacterium culture and allowed to dry.
Approximately 50 synchronized L1 worms were dispensed per plate, and kept at 20°C for 3 days
On the day of the tracking, 3 worms from each Agrobacterium plate were picked to a well of the prepared 96 well low peptone NGM agar plate.
Tracking
Tracking
Videos were acquired and processed as described in https://doi.org/10.1038/s42003-022-03206-1
Videos were acquired at 25 frames per second using a shutter time of 25 ms and a resolution of 12.4 µm/px. Three videos were taken sequentially: a 5 min pre-stimulus video, a 6 min blue light recording with three 10 s blue light pulses starting at 60, 160, and 260 s, and a 5 min post-stimulus recording. Videos were segmented and tracked using Tierpsy Tracker (https://doi.org/10.1098/rstb.2017.0375).
Following tracking, features were extracted describing morphology, “length_50th” (median worm length), and movement, “speed_50th” (median worm speed), “motion_mode_forward_frequency” (frequency of forward motion in events/second) and “motion_mode_pasued_frequency” (frequency of paused motion in events/second).