Additional file 1 of An optimized growth medium for increased recombinant protein secretion titer via the type III secretion system
Creators: name: Burdette, Lisa Ann
name: Wong, Han Teng
name: Tullman-Ercek, Danielle
Publisher: name: figshare
Year: 2021
Subjects: subject: Biophysics
subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Biotechnology
subject: Science Policy
subject: 69999 Biological Sciences not elsewhere classified
subject: 80699 Information Systems not elsewhere classified
subject: FOS: Computer and information sciences
subject: FOS: Computer and information sciences
subject: Developmental Biology
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: Plant Biology
subject: Computational Biology

description: Additional file 1: Table S1. Strains used in this study. Table S2. Plasmids used in this study. Table S3. Primers used in this study. Figure S1. A Relative expression (i) and representative western blots (ii) of SptP-DH-2xFLAG-6xHis in LB-L, 2X YT, and TB. B Relative expression (i) and representative western blots (ii) of SptP-DH-2xFLAG-6xHis in LB-L supplemented with the defined components in TB. All western blots are representative of four biological replicates. Samples were diluted to fall within the linear range of the LB-L signal. Boxed bands are from the same blot but were rearranged for clarity. “WCL” is whole culture lysate and “SF” is secreted fraction. Error bars represent standard error of the mean for four biological replicates. Table S4. Conductivity of media at start of experiment and OD600nm and secreted fraction pH at time of harvest. Figure S2. Western blots representative of three biological replicates for LB-L supplemented with various carbon sources. “WCL” is whole culture lysate and “SF” is secreted fraction. Table S5. OD600nm and secreted fraction pH at time of harvest for LB-L supplemented with various carbon sources. Figure S3. Representative histograms of flow cytometry GFP signal for SPI-1 transcriptional activity in LB-L with 0.4% w/v carbon sources (Fig. 2). Overlaid populations represent hourly time points from 1 h (red) to 8 h (teal). “ASTE13 WT” is a negative control. Figure S4. A Spearman correlations between expression or secretion per cell and hour at maximum mean fluorescence from flow cytometry in Fig. 2. B Spearman correlations between expression or secretion per cell and maximum mean fluorescence from flow cytometry in Fig. 2. Expression and secretion per cell were calculated by dividing densitometry by OD600nm and normalizing to LB-L with no additives. Each data point is a replicate. Table S6. Medium additives to evaluate the effect of phosphate buffer on expression and secretion titer. Species in each “level” were designed to have a similar buffer concentration and/or conductivity to the corresponding concentration of KH2PO4/K2HPO4. Figure S5. A Relative bulk expression of SptP-DH-2xFLAG in LB supplemented with the buffers and salts listed below the graph. Species in each “level” were designed to have a similar buffer concentration (MOPS, MOPS + NaCl) and/or conductivity (NaCl, MOPS + NaCl) to the corresponding concentration of KH2PO4/K2HPO4. Each column of the table corresponds to the group of bars above it, and each row of the column corresponds to a separate bar in the group. Data was normalized to LB-L using semi-quantitative western blotting. Error bars represent standard error for four biological replicates except for black-outlined bars, which represent six biological replicates. LB-L and LB-IM are denoted by dashed lines. B Relative bulk expression versus conductivity for the media in part A. The gray shading represents a 95% CI on the linear regression. Each data point is a replicate except for LB-L, which is labeled as a reference. C Western blots representative of four biological replicates. “WCL” is whole culture lysate and “SF” is secreted fraction. Table S7. Conductivity of media at start of experiment and OD600nm and secreted fraction pH at time of harvest for LB supplemented with buffers, salts, and carbon sources. Figure S6. A Relative bulk expression of SptP-DH-2xFLAG-6xHis in media containing 10 g/L tryptone, 5 g/L yeast extract, and the additives listed in Table S7. Bulk expression was normalized to LB-L with no additives (dotted line). Error bars represent standard error of the mean for three biological replicates. B Western blots are representative of three biological replicates. Samples were diluted to fall within the linear range of the LB-L signal. Boxed bands are from the same blot but were rearranged for clarity. “WCL” is whole culture lysate and “SF” is secreted fraction. Figure S7. Representative histograms of GFP signal for flow cytometry to measure SPI-1 transcriptional activity in the media listed in Table S7 with no added carbon source (“none”; Fig. 5). Overlaid populations represent hourly time points from 1 h (red) to 8 h (teal). “ASTE13 WT” is a negative control. Figure S8. Representative histograms of GFP signal for flow cytometry to measure SPI-1 transcriptional activity in the media listed in Table S7 with 0.4% w/v glucose (“glucose”; Fig. 5). Overlaid populations represent hourly time points from 1 h (red) to 8 h (teal). “ASTE13 WT” is a negative control. Figure S9. Representative histograms of GFP signal for flow cytometry to measure SPI-1 transcriptional activity in the media listed in Table S7 with 0.4% w/v glycerol (“glycerol”; Fig. 5). Overlaid populations represent hourly time points from 1 h (red) to 8 h (teal). “ASTE13 WT” is a negative control. Figure S10. A Spearman correlations between expression or secretion per cell and hour at maximum mean fluorescence from flow cytometry in Fig. 5. B Spearman correlations between expression or secretion per cell and maximum mean fluorescence from flow cytometry in Fig. 5. Expression and secretion per cell were calculated by dividing densitometry by OD600nm and normalizing to LB-L with no additives. Each data point is a replicate. Figure S11. A Relative bulk expression of test proteins from WT and ∆sipD strains in LB-L or LB-ES media. “LB-ES” is 10 g/L tryptone, 5 g/L yeast extract, 80 mM KH2PO4/K2HPO4 pH 7.4, 90 mM NaCl, and 0.4% w/v glycerol. Relative bulk expression was normalized to secretion from ASTE13 WT in LB-L for each protein using semi-quantitative western blotting. Error bars represent standard error of the mean for three biological replicates. B Western blots are representative of three biological replicates. Samples were diluted to fall within the linear range of the normalization signal. Boxed bands are from the same blot but were rearranged for clarity. “WCL” is whole culture lysate and “SF” is secreted fraction. Figure S12. Titer of secreted proteins.