Additional file 1 of Engineering a suite of E. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccines
Creators: name: Kay, Emily J.
name: Mauri, Marta
name: Willcocks, Sam J.
name: Scott, Timothy A.
name: Cuccui, Jon
name: Wren, Brendan W.
Publisher: name: figshare
Year: 2022
Subjects: subject: Microbiology
subject: FOS: Biological sciences

description: Additional file 1: Table S1. Plasmids used in this study. Table S2. Strains used in this study. Table S3. N20 specific sequence of gRNA and protospacer adjacent motif (PAM). Table S4. Primers for mutant confirmation. Table S5. 2 way ANOVA multiple comparisons for TLR4 activation assay. Figure S1. PCR Verification of mutations within strains. Overnight cultures of each strain were lysed using Chelex 100 (BioRad). PCR was performed with Platinum Green Hot start PCR mastermix (Invitrogen), in 20 µl reaction, according to manufacturer’s instructions, using Tm determined by NEB Tm calculator. Ten µl was run on an agarose gel alongside Hyperladder 1 Kb marker (Bioline) and visualized with gelRed nucleic acid stain (biotium). M marker; B negative control no template PCR. Figure S2. Growth curve of all strains. Overnight cultures of all strains were used to inoculate fresh LB broth to an OD600 of 0.03. Cultures were incubated at 37 °C with shaking and OD600 measurements taken at regular intervals with a final reading taken at 24 h. Strains did not contain plasmids. Strains depicted in red contain pglB and grow slightly slower than those without and reach a slightly lower final OD600. W3110 is included as a wild type comparison. The experiment was performed in triplicate and the data shown are mean values with error bars depicting standard error of the mean. Figure S3. Colony forming units for TLR4 activation assay. Overnight cultures of W3110, Raptor and ClearColi strains were matched for OD600 value, serially diluted in PBS, and plated on LB agar plates in triplicate. The plates were grown at 37 °C overnight before counting and determining CFU/ml. The average results were plotted, and a one-way ANOVA with Tukey’s multiple comparison test was used to determine whether the difference was significant. The OD matched samples were then further diluted in HEKblue cell culture media prior to the TLR4 activation assay. Figure S4. Frame shift in wzE within the pTarget recombination construct ΔwzzB(wzD-wzE). Chromatogram of sequence viewed in Chromas software (Technelysium Pty Ltd., Australia) with the base after deletion highlighted. Alignment of the pTarget recombination construct sequence (top) against the S. pneumoniae TIGR4 genome sequence (bottom) with ATG start codon of wzE in green. A thymine (T) is missing from the pTarget recombination construct sequence which causes a frame shift in the open reading frame of wzE. Figure 5. Conjugation of C. jejuni heptasachcaride to AcrA. Overnight culture was used to seed 2YP broth to OD600 of 0.03. After 4 h 1 mM IPTG was added to W311B pPgl::pglB pWA2. After 24 h growth at 28 °C cells were harvested, matched to OD600 20 and lysed with FastPrep homogenizer. Fifteen µl lysate was loaded on a Bolt 4–12% bis-tris gel with MOPS buffer. After transfer to nitrocellulose membrane protein and glycan were detected using mouse anti-His monoclonal antibody (Abcam, UK) and HR6 antiserum (S. Amber and M. Aebi, unpublished data) respectively. Secondary goat anti-rabbit IgG IRDye 800 and goat anti-mouse IgG IRDye 680 conjugates were used tp generate fluorescent signal which was detected using an Odyssey LI-COR detection system (LI-COR Biosciences UK Ltd.). Green channel is for Glycan, Red for protein. All strains contained pACYCPgl::pglB and pWA2. CLM24 does not contain a functional PglB transferase so only unclycosylated AcrA is visible. W311B contains chromosomally integrated pglB under a Ptac promoter and the remaining strains contain integrated pglB under an Anderson X10 promoter. M marker (PageRuler plus prestained protein ladder—Fisher). AcrA runs at 40 KDa.