Nucleobond pc 500 kit

Plasmids were purified from sorted UTI89/pMWLib using NucleoBond PC 500 kit (Macherey-Nagel) as specified by the manufacturer. PCR amplification of the inserted chromosome segments in pMWLib was performed using primers P1 (5′-TGAAGGCTCTCAAGGGCATC-3′) and P2 (5′-GTGTTGGCCATGGAACAGGT-3′). For Illumina sequencing, the DNA fragments were sonicated into 250–400 bp long segments from the original size of 500–700 bp, and sequencing library preparation was performed according to the manufacturer’s instructions (Illumina) as described previously [25 (link)]. The library was sequenced using the Illumina HiSeq 1500 instrument.
Reads were aligned using STAR version 2.3.1z_r395 with spliced mapping disabled. Subsequently, uniquely aligning reads overlapping operon promoters were counted, and DESeq2 was used to identify promoters that were utilized differentially in the urine sample. The rLogFC was calculated. A positive rLogFC means that more reads map to a particular operon promoter region in the sample grown in human urine compared to LB. The opposite is true of a negative score. Operon promoters were defined as the region spanning from 300 bp upstream of the transcription start site to 50 bp downstream. Operons were defined as bookended transcripts, i.e. genes with no base pairs separating them.

The human p53 isoform constructs used in this report were previously described (Bourdon et al., 2005 (link)). They were sub-cloned into the EcoRI and BamHI sites of pEGFPC1 (Clonetech) to obtain GFP-tagged proteins or in the BamHI and EcoRI sites of pLPCmyc to obtain myc-tagged proteins. Constructs were amplified using the Nucleobond PC 500 kit (Macherey-Nagel) according to the manufacturer’s instructions. Human Δ133p53 isoforms (α, β and γ) were cloned into pMSCVhyg (Clontech Laboratories) plasmid for retrovirus production. pSIREN-Luc (luciferase)-shRNA was purchased from Clontech. The mouse anti-E-Cadherin (clone 36) and the mouse anti-Beta1-Integrin, were purchased from BD-transduction laboratories and were diluted at 1/400° and 1/250°, respectively. The mouse N-Cadherin (clone 32/BD Transduction laboratories), and the rabbit polyclonal anti-GFP (Invitrogen, Life Technologies, A-6455) were used at 1/400°, 1/250°, 1/300° and 1/2000°, respectively.
The sheep pantropic p53 antibody Sapu was diluted at 1/5000°. The rabbit polyclonal KJC8 antibody specific of the β p53 isoforms was diluted at 1/4000° (Bourdon et al., 2005 (link)). The Horse-Radish Peroxidase (HRP)-conjugated anti-IgG antibodies were purchased from GE-Healthcare and were diluted at 1/5000°. The Western Lightning Chemiluminescence (ECL) reagents were purchased from PerkinElmer.

The sequence of StxB-scFv UCHT1 was custom-ordered in a pET22b+ plasmid (BioCat GmbH, Heidelberg, Germany). The chemically competent BL21(DE3) E. coli strain (New England Biolabs, Ipswich, MA, USA) was used for transformation, following the manufacturer´s instructions. The bacteria were briefly thawed on ice, and then 10 ng of DNA was added. The mix was incubated for 30 min at 4 °C. Afterwards, the reaction was stopped by heat shocking the bacterial mix for 20 s at 42 °C. Following this step, 350 µL of sterile LB-medium was added and incubated at 37 °C for 1 h under agitation (180 rpm). The solution was streaked out on LB agar plates containing ampicillin. The plates were kept overnight at 37 °C.
Following the incubation time, a single colony was picked and transferred to a tube with LB-medium. The culture grew overnight at 37 °C under continuous shaking (180 rpm). Afterwards, 100 mL of LB-medium was inoculated with the overnight culture, in the same conditions as mentioned above. The plasmid DNA of the bacteria was then purified (NucleoBond PC 500 kit, Macherey Nagel, Düren, Germany). The plasmid map of the resulting plasmid can be seen in Supplementary Figure S2.