Figure 7.

Western blot analysis of the GFP in strain CSMI21 and control strains. The Anabaena strains used were PCC 7120 (WT), mutant CSMI21 (alr2310-gfp) and a strain carrying pAM1954, which is a replicative plasmid bearing the gfp gene expressed from the rbc gene promoter. Cell-free extracts were prepared from whole filaments grown in bubbled cultures with BG11 medium or incubated in BG11₀ medium for 48 h, or from heterocysts (Hets) isolated from the latter. Anabaena sp. [pAM1954] was grown in shaken cultures containing BG11 medium. Proteins were subjected to SDS-PAGE electrophoresis and transferred to membranes. Protein detection was performed with anti-GFP antibodies. Native GFP is 26.9 kDa and the Alr2310-GFP fusion, which includes Alr2310, a tetra-glycine linker peptide, and the GFP is 65.7 kDa. Protein loaded was 40 μg per well (0.6 μg in the case of Anabaena [pAM1954]). As observed with the wild-type extracts, the antibodies marked 3 or 4 unspecific bands (brackets). Strain CSMI21 produced mainly a band corresponding to the fusion protein (black arrow) and only a faint band corresponding to free GFP, which was identified with the extract of Anabaena [pAM1954] that only produced, as expected, free GFP (open arrow). Note that some material from lane BG11/CSMI21 may have contaminated lane BG11/WT.