Difference between revisions of "Part:BBa K5136028"

Revision as of 21:21, 1 October 2024

sfgfp

Biology

Superfolder GFP (sfGFP) is a superfolding derivative of green fluorescent protein (GFP), an optimized green fluorescent protein obtained through modifications to the wild-type GFP. It’s reported that sfGFP exhibits significant advantages over conventional GFP in several aspects, including folding efficiency within cells, fluorescence maturation rate, and brightness (1, 2).

Usage and design

Superfolder GFP is primarily applied in our autolytic system. We utilized the E. coli autolytic system FLSA/LLSA (FhuD/LMT-lysozyme-SsrA mediated autolytic system), where the signal peptide directs T7 lysozyme to the peptidoglycan layer to promote cell lysis, and the SsrA tag ensures the degradation of the T7 lysozyme that leaks into the cytoplasm.
To validate the efficacy of the autolytic system, specifically its cell lysis and protein release capabilities, we selected sfGFP as the reporter. We co-transformed a plasmid containing sfGFP with a plasmid carrying the FLSA/LLSA autolytic module into engineered bacteria. The intensity of the fluorescent protein reflects the amount of protein inside and outside the cells. By measuring the ratio of the fluorescence intensity in the supernatant to the total fluorescence intensity, we quantitatively analyze the lysis efficiency, thereby evaluating the performance of the autolytic system.

Characterization

sfGFP Release Efficiency Determination

After co-transforming the plasmid pSB1C3 containing the FLSA/LLSA coding system and sfGFP_pET-28a(+) into E. coli BL21 (DE3), the cultures were grown overnight in LB medium containing corresponding antibiotics. The cultures were diluted and grown to OD₆₀₀ 0.6-0.8, followed by the addition of 0.5 mM IPTG to induce sfGFP expression at 18°C. After 10 hours, 0.25% L-arabinose was added to activate the autolytic system. The total fluorescence intensity was measured after 16 h of expression of the induced autolysis system, and after centrifugation, the fluorescence intensity of the supernatant was measured too. The ratio of the fluorescence intensity of the culture and supernatant was used to assess the lysis efficiency of FLSA system.

   By using sfGFP as the reporter, we successfully determined the lysis efficiency of four autolytic systems, providing strong support for the efficient release of our subsequent target enzymes.

Reference

1. T. Dinh, T. G. Bernhardt, Using superfolder green fluorescent protein for periplasmic protein localization studies. J Bacteriol 193, 4984-4987 (2011).
2. J. Shen et al., Strategies to improve the fluorescent signal of the tripartite sfgfp system. Acta Biochim Biophys Sin (Shanghai) 52, 998-1006 (2020).

Sequence and Features