Sep 09, 2017
- 1Laboratorio de Tecnologías Libres, iBio;
- 2Pontificia Universidad Católica de Chile
- Laboratorio de Tecnologias Libres
External link: https://doi.org/10.1371/journal.pone.0187163
Protocol Citation: Isaac Núñez, Tamara Matute 2017. Colony-sectoring assay. protocols.io https://dx.doi.org/10.17504/protocols.io.jsecnbe
Manuscript citation:
Nuñez I, Matute T, Herrera R, Keymer J, Marzullo T, Rudge T, Federici F (2017) Low cost and open source multi-fluorescence imaging system for teaching and research in biology and bioengineering. PLoS ONE 12(11): e0187163. doi: 10.1371/journal.pone.0187163
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
Created: September 08, 2017
Last Modified: March 20, 2018
Protocol Integer ID: 7718
Abstract
This protocol describes the procedure to obtain single bacterial colonies composed of multiple sectors of different strains. Each sector is originated from different founder cells (situated in close proximity to each other at the moment of plating). This protocol is based on Hallatschek et al. 2007 (Hallatschek O, Hersen P, Ramanathan S, Nelson DR. Genetic drift at expanding frontiers promotes gene segregation. Proc Natl Acad Sci U S A. 2007 Dec 11;104(50):19926-30).
This assay has been used for low cost and open source fluorescence imaging (please see https://osf.io/dy6p2/ for further information & data, https://github.com/SynBioUC/FluoPi for code and http://docubricks.com/viewer.jsp? for hardware assembly)
Materials
MATERIALS
P2 micropipette and tips
P20 micropipette and tips
P200 micropipette and tips
LB agar plates with the proper antibiotic (e.g. Kanamycin)
1.5 ml tubes to perform the dilutions
Shaker incubator
Sterile conditions (e.g. laminar flow or a flame)
14 ml round bottom culture tubes
Growth
Growth
Grow independent liquid cultures of the bacterial strains (e.g. three bacterial cultures: E.coli Top10 with CyOFP expression plasmid, mBeRFP expression plasmid and sfGFP expression plásmid) overnight in 5 ml LB media with the proper antibiotic (e.g. Kanamycin for the plasmids mentioned above) at 37°C with agitation.
Mixing
Mixing
Mix all cultures proportionally (eg, 100 μl of bacterial culture containing the CyOFP plasmid, 100 μl of bacterial culture containing the mBeRFP plasmid and 100 μl of bacterial culture containing the sfGFP plasmid).
Dilution
Dilution
Dilute the mixture from step 2 in order to start the colony with fewer cells and number of sectors. It is recommended to dilute 1: 100 (top right image) or 1: 10,000 (bottom right image) with LB media and place a drop of 0.5 ul in the center of a LB agar plate with corresponding antibiotics (e.g. Kanamycin for the plasmids used here).
Plating
Plating
Take 0.3 μl of the previous step and place it in the center of a LB agar plate with the proper antibiotic(s) in common of the strains (e.g. Kanamycin).
Incubation
Incubation
Incubate the plate at room temperature for a period of 2 to 10 days (or 37°C for a shorter period of time). Take into account that the incubation temperature is a key determinant of colony growth rate.