organism: Escherichia coli O17 str. K12a
description: Protocols: RNA extraction and RNAseq have been performed in two different batches, corresponding to Ag43 and the nanobodies experiments. Bacteria were grown as described above until OD600 =0.5 and then left for aggregation at 37°C for 3h in separating funnel. Then, by opening the tap of the separating funnel, only the lower part of the culture, corresponding to the aggregated part (in strain forming aggregates), is collected and placed in an eppendorf tube. The aggregated or non aggregated cells were then washed with 2 volumes of RNAprotect (Qiagen) to prevent RNA degradation, and the pellets were kept at -80°C Bacterial strains used in this study are listed in Supplementary table 8 S8. All strains were grown in Miller's Lysogeny Broth (LB) (Corning) supplemented with chloramphenicol (25µg/mL), kanamycin (50µg/mL) or ampicillin (100µg/mL) when needed. Cultures where incubated at 37°C with 180 rpm shaking during growth phase or put at 37°C in static condition during aggregation phase. Cultures on solid media were done on LB with 1.5% agar supplemented with antibiotics when required. Prior to liquid cultures, bacteria were always streaked on LB agar from glycerol stocks. All media and chemicals were purchased from Sigma-Aldrich unless mentioned otherwise. All experiments and genetic construction were done in E. coli K12 MG1655 (F-,-, rph-1) obtained E. coli genetic stock center CGSC#6300. Ag43 (Flu) was used as a “natural” adhesin since it is the major aggregation factor in E. coli K12. For the Ag43 natural adhesin, we used two sets of couples. The first couple is composed of ∆flu deleted for ag43 and its isogenic opposite, PcLflu, constitutively expressing Ag43 (high level of Ag43). Since Ag43 is a naturally phase variable adhesin we also used a strain in which the Ag43 ON or OFF status is traceable through the use of an operon fusion of ag43 with the lacZ gene. The second couple is therefore composed of cultures of “lacZ blue” Ag43 ON cells and “lacZ white” Ag43 OFF cells. For the synthetic adhesins, we used “chimeric” proteins recently described in (Glass and Riedel-Kruse, 2018). These fusion proteins mainly consist of an anchoring and an adhesin domain. The outer membrane anchor is composed of the intimin N terminus from an enterohemorrhagic E. coli (EHEC O157:H7), including a small N-terminal signal peptide, a LysM domain for peptidoglycan binding, and a β-barrel for transmembrane insertion. The adhesin domain, composed of a nanobody (the variable domains of camelid heavy-chain antibodies) or its corresponding antigen, is then fused to the C-terminus part of the truncated intimin. We introduced the corresponding genetic constructions to replace the ag43 gene, under the control of the constitutive lambda PR promoter PcL. We used four couples of synthetic adhesins: Nb2-Ag2, Nb3-Ag3, Nb3-Ag2 and Nb2-Ag3. 1:1 mixture of cells constitutively expressing Ag2 and Nb2, and Ag3 and Nb3 were used as synthetic adhesins aggregates, while 1:1 mixture of cells constitutively expressing Ag2 and Nb3, and Ag3 and Nb2 were used as synthetic adhesins non-aggregating cells. E. coli MG1655 has the F-,l-, rph-1 genotype. ∆flu = MG1655 deleted of agn43, KmR. MG1655 PcLflu λPr placed in front of agn43, resulting in constitutive expression of the gene, CmR. MG1655 EndfluLacZzeo = lacZ gene is transcriptionally fused at the end of agn43, allowing the tracking of the ON/OFF status of the natural promoter of agn43, ZeoR. All other genotypes mean the synthetic adhesion locus is placed at the agn43 locus site, under the control of the λPr, KmR, CmR. Total RNA was extracted using MP Biomedicals FastRNA ProTM BLUE KIT following provider's manual and treated with Ambion Turbo DNA-free™ kit to remove possible DNA contamination. Total RNA from 4 independent replicates for each strain/couple were checked on RNA6000 RNA chips on Bioanalyzer (Agilent) for its quality and integrity. The QIAseq Fast Select -5s/16s/23s (QIAGEN) was used for ribosomal RNA depletion according to manufacturer instructions. Libraries were built using a TruSeq Stranded mRNA library Preparation Kit (Illumina, USA) following the manufacturer's protocol. Quality control was performed on an Agilent BioAnalyzer.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-12-31
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-12-31
attributes.tag: External Id
attributes.value: SAMEA13318181
attributes.tag: INSDC center alias
attributes.value: 1. Plate-forme Technologique Biomics – Centre de Ressources et Recherches Technologiques (C2RT), Institut Pasteur, Paris, France 2. Hub de Bioinformatique et Biostatistique - Departement Biologie Computationnelle, Institut Pasteur, 75015 Paris, France
attributes.tag: INSDC center name
attributes.value: 1. Plate-forme Technologique Biomics – Centre de Ressources et Recherches Technologiques (C2RT), Institut Pasteur, Paris, France 2. Hub de Bioinformatique et Biostatistique - Departement Biologie Computationnelle, Institut Pasteur, 75015 Paris, France
attributes.tag: INSDC first public
attributes.value: 2022-12-31T00:57:52Z
attributes.tag: INSDC last update
attributes.value: 2022-12-31T00:57:52Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11396:LacZ_Bleue_2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: genotype
attributes.value: MG1655 EndfluLacZzeo - ON
attributes.tag: growth condition
attributes.value: LB agar
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: whole organism
attributes.tag: sample name
attributes.value: E-MTAB-11396:LacZ_Bleue_2
attributes.tag: strain
attributes.value: MG1655