Real-Time Quantification of the Effects of IS200/IS605 Family-Associated TnpB on Transposon Activity.
J Vis Exp
; (191)2023 01 20.
Article
em En
| MEDLINE
| ID: mdl-36744795
ABSTRACT
Here, a protocol is outlined to perform live, real-time imaging of transposable element activity in live bacterial cells using a suite of fluorescent reporters coupled to transposition. In particular, it demonstrates how real-time imaging can be used to assess the effects of the accessory protein TnpB on the activity of the transposable element IS608, a member of the IS200/IS605 family of transposable elements. The IS200/IS605 family of transposable elements are abundant mobile elements connected with one of the most innumerable genes found in nature, tnpB. Sequence homologies propose that the TnpB protein may be an evolutionary precursor to CRISPR/Cas9 systems. Additionally, TnpB has received renewed interest, having been shown to act as a Cas-like RNA-guided DNA endonuclease. The effects of TnpB on the transposition rates of IS608 are quantified, and it is demonstrated that the expression of TnpB of IS608 results in ~5x increased transposon activity compared to cells lacking TnpB expression.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Bactérias
/
Elementos de DNA Transponíveis
Tipo de estudo:
Risk_factors_studies
Idioma:
En
Revista:
J Vis Exp
Ano de publicação:
2023
Tipo de documento:
Article