Screening Protein Interactions in E. coli

Plasmids encoding fusions of glycosyltransferases and proteins involved in the transport and polymerization of EPS (PssT, PssP, PssL, and PssP2) were previously constructed [26 (link),76 (link),78 (link)] and are listed in Supplementary Table S8. Plasmids were co-transformed into E. coli DHM1 strain and interaction screening was performed using agar plates containing 40 μg/mL 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal, A&A Biotechnology, Gdańsk, Poland), 0.5 mM IPTG (A&A Biotechnology, Gdańsk, Poland), ampicillin, and kanamycin. Quantitative measurement of β-galactosidase activity was performed in a plate format as described earlier [26 (link)].

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Żebracki K., Horbowicz A., Marczak M., Turska-Szewczuk A., Koper P., Wójcik K., Romańczuk M., Wójcik M, & Mazur A. (2023). Exopolysaccharide Biosynthesis in Rhizobium leguminosarum bv. trifolii Requires a Complementary Function of Two Homologous Glycosyltransferases PssG and PssI. International Journal of Molecular Sciences, 24(4), 4248.

Publication 2023

IPTG

Agar Ampicillin E coli Galactoside Glycosyltransferases Iptg Kanamycin Plasmids Polymerization Proteins Strain X gal β galactosidase

Corresponding Organization : Maria Curie-Skłodowska University

Top 5 similar protocols

Variable analysis

independent variables

Plasmids encoding fusions of glycosyltransferases and proteins involved in the transport and polymerization of EPS (PssT, PssP, PssL, and PssP2)

dependent variables

Interaction screening using agar plates containing 40 μg/mL 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal)
Quantitative measurement of β-galactosidase activity

control variables

IPTG concentration (0.5 mM)
Ampicillin
Kanamycin

Annotations

Based on most similar protocols

To optimize experimental performance, the protocols suggest co-transforming plasmids into the E. coli DHM1 strain (Protocol 1, Input protocol).

The protocols use agar plates containing 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal) and isopropyl-β-D-galactoside (IPTG) as a colorimetric assay to screen for protein-protein interactions (Protocol 1, Input protocol).

Quantitative measurement of β-galactosidase activity, an indicator of protein-protein interactions, is performed in a microplate or plate format as described in the earlier references (Protocol 1, Input protocol).

The protocols use ampicillin and kanamycin as selection markers for the plasmids (Protocol 1, Input protocol).

The plasmids encoding fusion proteins or components of the Wzx/Wzy-dependent transport and polymerization system of EPS (PssT, PssP, PssL, and PssP2) are listed in the supplementary tables of the referenced publications (Protocol 1, Input protocol).

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