Fig. 5
Automated screening comparing glycoconjugate expression across multiple strain variants cultured in parallel on the automated platform. a–b Comparison across five E. coli strains and a negative control that does not express PglB; a Standard and overexposed western blotting showing the presence of ExoA-CPS4 glycoproteins in periplasmic extracts and the subsequent densitometry analysis of the blots based on pixel intensity normalised to a control condition; b Sandwich ELISA analysis of periplasmic extracts shown in a. Dashed line showing background CPS4 glycan signal threshold from negative control strain without PglB; c–e Comparison across three strain variants with and without induction of PglB expression; c Western blotting and subsequent densitometry analysis; d Sandwich ELISA analysis of samples shown in c; e OD600 of Falcon (Strain 6) cultures, measured pre-induction and at harvest; f–g Comparison of Falcon strain expressing CPS4, PglB (or empty backbone for the negative control) and carrier protein ExoA with either 2 or 10 glycosylation sequons from either a pEC415 or pEXT20 plasmid backbone. Expression under a DsbA or PelB signal peptide was also compared; f Western blotting and subsequent densitometry analysis; g Sandwich ELISA analysis of extracts shown in f. Dashed line showing background CPS4 glycan signal threshold from negative control strain lacking PglB. Statistical significance (*) using an unequal variance two-tailed t-test is considered when p ≤ 0.05