Additional file 1 of Nanobodies as potential tools for microbiological testing of live biotherapeutic products

Additional file 1:Table S1. Bacterial strains, plasmids, and primers used in this study. Figure S1. ELISA results of the interaction between different lactobacilli and secreted nanobodies. (A) Lc58 and Lc38 nanobody interaction between target L. crispatus antigen (strains 33820 and 33197) and control lactobacilli antigens. (B) Lj94 and Lj75 nanobody interaction between target L. jensenii JV-V16 antigen and control lactobacilli antigens. Secreted nanobody concentrations were evaluated using Octet (Sartorius) and the preparations were diluted to 1 μg/ml for experiments. Figure S2. SDS PAGE gels showing purified nanobodies and fluorescently tagged nanobodies. Proteins were loaded on NuPAGE 4–12% BisTris gels and stained with Coomasie Blue. The expected molecular mass of each protein and the lane in which the purified protein was run is indicated in the boxes below the gels. Molecular weight markers are identified on the left. Please note that under boiling SDS conditions, TagRFP is known to fragment. The additional bands observed in (B) lane C are likely due to the fragmentation of sample preparation for SDS PAGE. Figure S3. L. jensenii 115-3-CHN Lj75 antigen identification. (A) AA sequence analysis of (1) the originally annotated AA sequence of L. jensenii 115-3-CHN antigen (EEX23860.1), (2) the confirmed L. jensenii JV-V16 Lj75 antigen AA sequence, and (3) the extended L. jensenii 115-3-CHN Lj75 antigen AA sequence. Green above sequence analysis indicates 100% AA sequence identity. (B) Depiction of unique peptide hits along the AA sequence of the corrected L. jensenii 115-3-CHN antigen sequence. Green indicates where in the AA sequence the unique peptides match. Figure S4. L. crispatus strain lysate western blots with Lc58. L. crispatus EX8 VC07 (Lane 1), L. crispatus 125-2-CHN (Lane 2), or L. jensenii 25258 (lane 3) lysates were probed with Lc58. Lc58 binding was detected with an anti-his HRP conjugated secondary antibody. Figure S5. Detection of nanobody target candidate expression by western blot with HRP conjugated anti-FLAG antibody probing. (A) Lc58 target candidates; Lane 1, L. crispatus 125-2-CHN lysate; Lane 2, empty vector; Lane 3, S-layer (EEU18441.1) ; Lane 4, Bacterial Ig-domain protein (EEU19392.1) ; Lane 5, Cell separation protein (EEU18637.1). (B) Lj75 candidates; Lane 1, L. jensenii JV-V16; Lane 2, empty vector; Lane 3, NlPC/P60 family protein (EFH30000.1); Lane 4, Hypothetical protein (EFH30544.1).Figure S6. Use of SYTOX Green Ready Flow reagent to distinguish live from dead cells. SYTOX (ThermoFisher) is a cell impermeant nucleic acid stain that enters cells with damaged membranes and binds nucleic acids. (A) Untreated and unstained L. crispatus 33820, (B) Untreated L. crispatus 33820 solution (prepared same as flow cytometry samples), and (C) Isopropyl alcohol treated (70%, 25 min)L. crispatus 33820. (D) Untreated and unstained L. jensenii 115-3-CHN, (B) Untreated L. jensenii 115-3-CHN solution (prepared same as flow cytometry samples), and (C) Isopropyl alcohol treated (70%, 25 min.) L. jensenii 115-3-CHN. Please note that GFP and AlexaFluor use same laser and filter settings on the flow cytometer used in this assay