Journal: STAR Protocols

Article Title: Protocol to identify the signaling network of nucleotide second messengers in Shigella sonnei

doi: 10.1016/j.xpro.2026.104353

Figure Lengend Snippet: Graphical depiction illustrating regulation on the transcription of a c-di-GMP synthase encoding gene by the cAMP signaling system (Top) The S. sonnei CMCC51592 wild-type and crp mutant strains were incubated in LB medium at 37 °C until the OD 600 was 0.8. Then, 1.0 mL of the cultured bacterial suspension was transferred into a new 1.5 mL microcentrifuge tube. Total RNA was extracted, and cDNA was synthesized via reverse transcription. Primers were designed based on genes encoding key c-di-GMP metabolic enzymes, and RT–qPCR was performed to determine the effect of crp deletion on the expression of these genes. (Bottom) Biotin was used as a marker to label the purified promoter DNA fragment at its 3′ end to create a probe sample. DNA–protein (with or without cAMP) binding reactions were prepared by coincubating biotin-labelled probes with proteins. The DNA–protein complexes were separated from the unbound probes using a 5% polyacrylamide gel. Biotin-labelled probes with different mobilities were detected on the membrane. Figure reprinted with permission from Wang et al., 2025. Created with BioRender.

Article Snippet: Biotin 3′ End DNA Labeling Kit , KeyGEN BioTECH , Cat#KGS132.

Techniques: Mutagenesis, Incubation, Cell Culture, Suspension, Synthesized, Reverse Transcription, Quantitative RT-PCR, Expressing, Marker, Purification, Binding Assay, Membrane