Abstract
Bacillus subtilis (B. subtilis) is a model Gram-positive organism used to study a variety of physiological states and stress responses, one of which is the development of competence. Competence refers to the physiological state of a cell which allows it to be transformed naturally. Through induction of competence, the efficiency of natural transformation can be quantified by plating colony forming units (CFU) and transforming units (TFU). Here we describe a protocol for quantifying relative transformability using B. subtilis.
Keywords: Natural transformation, Competence, Bacillus subtilis, Quantitative transformation efficiency assay, Horizontal gene transfer (HGT), DNA uptake
Background
Natural transformation is a type of horizontal gene transfer that involves a bacterium importing exogenous DNA from the environment and recombining that DNA into its genome. This process differs from induced transformation in that naturally transformable bacteria produce proteins that facilitate the import of DNA. Modification of the cell membrane, such as through electroporation or heat shock, is typically essential for induced transformation. Natural transformation can lead to the acquisition of new genetic elements, such as antibiotic resistance or catabolic genes, and may allow bacteria to adapt rapidly to their environment. The various proteins involved with the development of competence, or the physiological state of a cell which allows it to be transformed naturally, and the mechanism of natural transformation are not yet fully understood. Quantitative analysis of transformation efficiency differences between a mutant and wild type provides insight into the contribution and potential functions of the gene in question. Beginning with the description of requirements for transformation of laboratory strains of Bacillus subtilis by Anagnostopoulos and Spizizen (1961), numerous variations on the original media and conditions for transformation, both qualitative and quantitative methods, have been described including but not limited to those by Dubnau and Davidoff-Abelson (1971) and Cutting and Vander Horn (1990). The experimental procedure described here is similar to those prior, focusing on currently available and preferred materials (such as casein hydrolysate, which has been found to produce varying efficiencies based on lot and manufacturer), and includes description of statistical analysis of the results.
Materials and Reagents
Equipment
Procedure
Data analysis
Recipes
Acknowledgments
This protocol was adapted from Chilton et al. (2017). This work was funded in part by a grant from the Rita Allen Foundation.
Competing interests
The authors have no conflicts of interest or competing interests with respect to this work.
References
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