Fig. 2
The glycosyl-engineered E. coli chassis with genomic integration of acceptor protein SC4573 and glycosyltransferase PglL. a Schematic representation of the design of glycosyl-engineered E. coli chassis. b PCR validation of the expression cassette LacI-pglL-SpyCatcher4573-rrnB. c Growth curve of the chassis strain WdlO-tPS. Each strain was inoculated with a seed solution at OD600 = 2.0, using an LB ratio of 1:100. d Genetic stability testing. After 10 generations of passaging, 96 monoclonal clones were selected and tested for positivity of the SC4573 and pglL genes in strains WdlO-d01/pSC4573 and WdlO-tPS using PCR. These were tested three times, and the positivity of the SC4573 and pglL genes in strains WdlO-d01/pSC4573 were 66.6% (32/96), 60.5% (38/96), 76.1% (23/96), respectively, with a mean of 67.7%. (e) Detection of acceptor protein SC4573 expression in strain WdlO-tPS. f Detection of KPO1-SC and KPO2-SC glycoprotein expression in strain WdlO-tPS. g Purified KPO1-SC and KPO2-SC glycoproteins produced by strain WdlO-tPS were analyzed by SDS-PAGE, and WB with anti-His, anti-KPO1, and ant-KPO2 antibodies. h Comparison of KPO1-SC and KPO2-SC glycoprotein expression before and after deletion of the yfdGHI gene cluster in strain WdlO-tPS