New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria

Abstract

Use of the green fluorescent protein (Gfp) from the jellyfishAequorea victoria is a powerful method for nondestructive in situ monitoring, since expression of green fluorescence does not require any substrate addition. To expand the use of Gfp as a reporter protein, new variants have been constructed by the addition of short peptide sequences to the C-terminal end of intact Gfp. This rendered the Gfp susceptible to the action of indigenous housekeeping proteases, resulting in protein variants with half-lives ranging from 40 min to a few hours when synthesized in Escherichia coli andPseudomonas putida. The new Gfp variants should be useful for in situ studies of temporal gene expression.

Evaluating Light‐Induced Promoters for the Control of Heterologous Gene Expression in Synechocystis sp. PCC 6803

“…26 eYFP excites and fluoresces at wavelengths well positioned between the absorption wavelengths of many light harvesting pigments found within cyanobacteria. 27 The remainder of the constructs described in Table 1 were generated using Gibson Assembly. 28…”

Low-oxygen tensions found inSalmonella-infected gut tissue boostSalmonellareplication in macrophages by impairing antimicrobial activity and augmentingSalmonellavirulence

“…To construct the SPI-2 reporter plasmid pWRG358, a codonoptimized variant of SFGFP (Pedelacq et al, 2005) including the destabilizing C-terminal LVA tag (Andersen et al, 1998) was synthesized (Life Technologies). Subsequently, sfgfp::LVA was amplified by PCR using primers SmaI-RBS-SFGFP-for and LVA-NcoI-rev and the product was cloned via SmaI/NcoI in the similarly digested pWRG15 (Wille et al, 2012), yielding pWRG218.…”

Progression of bacterial infections studied in real time ? novel perspectives provided by multiphoton microscopy

“…Wild-type GFP is very stable with a half-life of at least 24 h in Escherichia coli (Bongaerts et al, 2002). Modifications of the C-terminal end of the GFP protein have, however, resulted in variants which are susceptible to the action of indigenous housekeeping proteases, resulting in GFP derivates with half-times as short as 40 min when synthesized in E. coli and Pseudomonas putida (Andersen et al, 1998).…”