Additional file 1 of Dynamic feedback regulation for efficient membrane protein production using a small RNA-based genetic circuit in Escherichia coli
Creators: name: Guidi, Chiara
name: De Wannemaeker, Lien
name: De Baets, Jasmine
name: Demeester, Wouter
name: Maertens, Jo
name: De Paepe, Brecht
name: De Mey, Marjan
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Space Science
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Computational Biology

description: Additional file 1: Figure S1. Growth curves corresponding to indirect stress detection tool. Figure S2. cpxQ binding sites for nhaB mRNA. Figure S3. Bar plots representing the output sfGFP/OD600, mKate2, sfGFP and µmax for the control plasmid (blue) and the plasmid expressing the sRNA cpxQmut1 (grey) and this for different L-arabinose concentrations. Figure S4. Confocal scanning light microscopy images with intensity cross-section profiles for both the red FM4-64 membrane dye (purple colour) and the green fluorescent protein signal of TMD-sfGFP fusion proteins. Figure S5. Evaluation of the three dual plasmid designs (D1-3) on growth and protein production of the membrane targeted sfGFP. Figure S6. Library of nine different cpxQ mutants in their secondary structure with the associated ΔG2 value. Figure S7. Effect of the smart library of sRNA-based stress circuits on fluorescence intensity. Figure S8. Relative mKate2/OD600 fluorescence of Escherichia coli (E. coli) DE3 cells producing either sfGFP (cytoplasmatic superfolder green fluorescent protein, negative control), NlpE (IMP of E. coli, positive control), SohB(TMD)-sfGFP, YidC-sfGFP and GarP-sfGFP for several IPTG-concentrations (Timepoint: 10h, stationary phase). Figure S9. Effect of the sRNA-based circuit, respectively with cpxQmut1 (Stress Circuit 1) and cpxQmut3 (Stress Circuit 3), on cell growth and fluorescence intensity. Figure S10. Confocal scanning light microscopy images with intensity cross-section profiles for both the red FM4-64 membrane dye (purple colour) and the green fluorescent protein signal of MPs YidC and GarP (sfGFP, green colour). Figure S11. Effect of the non-specific and specific sRNA-based stress circuit on protein production of functional MPs expression. Figure S12. Detailed plasmid maps of the vectors created in this work. Table S1. List of fold changes for NlpE and sfGFP expression (at OD600 = 0.3) measured for indirect IM stress analysis. Table S2. Bacterial Escherichia coli strains that were used in this study. Table S3. List of DNA sequences used in this study to design the smart small RNA library. Table S4. Two sample t-tests performed in this study. Table S5. Statistical one-way ANOVA performed in this study. Table S6. Plasmids that were used and constructed throughout this study. Table S7. List of proteins used in this study for membrane localisation and membrane targeting. Table S8. List of fold changes for NlpE, sfGFP, SohB(TMD)-sfGFP, YidC-sfGFP and GarP-sfGFP expression (at t = 20h) measured for indirect IM stress analysis. Table S9. Overview of the different plasmid backbones used in this study and their assigned function. Table S10. List of DNA sequences used in this study (coding sequences, promoter, 5’UTRs and terminator sequences).