Journal: Microbial cell factories
Article Title: Genetic engineering of E. coli K-12 for heterologous carbohydrate antigen production.
doi: 10.1186/s12934-025-02749-2
Figure Lengend Snippet: Fig. 1 Schematic map of suicide vectors. A All suicide vectors were derived from pHY093 (pRE112-Cm-GFP) or pHY094 (pRE112-Kan-GFP). B Two homologous arms, i.e., HA_up and HA_down were cloned from E. coli W3110 genome and fused together by fusion PCR. The fused homologous arms replaced the GFP cassette in pHY093 (pRE112-Cm-GFP), resulting in pHY095 (pRE112-Cm-ΔOAg). C the viaB_locus was cloned from S. Typhi genome. The linearized pHY095 (pRE112-Cm-ΔOAg) vector and viaB_locus were assembled in vitro, resulting in pHY129 (pRE112-Cm-ΔOAg::viaB locus). D A cassette of FRT-flanked chloramphenicol resistance gene was cloned from pCP3, and cloned into the middle of homologous arms, resulting in pHY099 (pRE112-Km-ΔOAg::Cm_FRT2). E The OAgST O-antigen gene cluster was cloned from S. Typhimurium genome via Long-range PCR. The linearized pHY099 (pRE112-Km-ΔOAg::Cm_FRT2) vector and OAgST were assembly in vitro, resulting in pHY100 (pRE112-Km-ΔOAg::OAgST-Cm_FRT2). F The OAgSE O-antigen gene cluster was cloned from S. Enteritidis genome via Long-range PCR. The linearized pHY099 (pRE112-Km-ΔOAg::Cm_FRT2) vector and OAgSE were assembly in vitro, resulting in pHY101 (pRE112-Km-ΔOAg::OAgSE-Cm_FRT2). G pHY102 (pRE112-Km-ΔOAg::OAgSA-Cm_FRT2) was derived from pHY101 by deliberately deleting the tyv gene
Article Snippet: The gene cluster insertion mutations on the E. coli chromosome were constructed using the sucrose counter-selectable pRE112 (addgene, plasmid #43,828) suicide vectors [24].
Techniques: Derivative Assay, Clone Assay, Plasmid Preparation, In Vitro, Long Range PCR
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