Part:BBa_K5237015

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This part can be used for the display of nanobodies on the surface of E.coli. Salema, V. et al, (2013) showed efficient presentation of nanobodies on the surface of E.coli K-12 cells by fusing them to the β domain of Intimin. The part comes from the pNeae2 plasmid (addgene #168300) and encodes an N-terminal signal peptide for Sec-dependent translocation into the periplasm, a LysM domain that interacts with the peptidoglycan and provides anchoring, and a β-barrel that inserts into the outer membrane. The C-terminus is exposed to the extracellular milieu and contains a myc tag (EQKLISEED) that can be used for purification or detection of full-length protein.

Figure 3: Fluorescence measurement of mNeonGreen, mScarlet-I and FRET. Fluorescence intensity of mNeonGreen (ex. 490 nm, em. 530 nm), mScarlet-I (ex. 560 nm, em. 600 nm), and FRET (ex. 490 nm, em. 600 nm) was measured 18 hours after IPTG induction (0.05 mM) and normalized to cell count (OD₆₀₀). Statistical significance was determined with Ordinary two-way ANOVA withŠidák's multiple comparison test, with a single pooled variance. *p < 0.05, ****p < 0.001. Data is depicted as mean (n=3) ± SD

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