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. Author manuscript; available in PMC: 2020 Jan 7.
Published in final edited form as: ACS Synth Biol. 2019 Jan 7;8(1):181–190. doi: 10.1021/acssynbio.8b00448

Figure 4.

Figure 4.

SpyCatcher protein fusions covalently bind to RsaA-SpyTag with variable occupancy according to the SpyTag location. (a) Flow cytometry histograms of RFP fluorescence per cell for strains expressing wild-type RsaA (black) and RsaA-SpyTag (colored lines) incubated with SpyCatcher-mRFP1 for 1 h. Baselines are offset for clarity. All eight strains displaying RsaA-SpyTag show an increase in the intensity of RFP fluorescence over the negative control with their intensity varying based on where SpyTag is inserted within RsaA. (b) SDS-PAGE of whole cell lysates from the rsaA467:SpyTag strain incubated for 24 h without (lane 2) and with (lane 3) SpyCatcher-mRFP1 protein. Appearance of a higher molecular weight band only in the reaction containing SpyCatcher-mRFP1 indicates covalent binding to RsaA-SpyTag.