Vibrio cholerae
Protocols: The Cre- and Xer-based SC2 reporters and the Cre-activity reporter were randomly inserted by transposition. Aliquots of the transposon library were thawed on ice and ~a billion cells were diluted into 100 ml of media supplemented with IPTG. Cells were grown until exponential phase (OD600 0.2). Then, ~a billion cells were centrifuged and transferred into 10 ml of fresh media without IPTG. After 30 minutes, arabinose was added at a final concentration of 0.02%. ~109 cells were recovered and frozen in liquid nitrogen at different time points after induction. For the replication arrest assay, rifampicin (300 µg/ml) was added 10 minutes after the induction of the expression of the recombinase. Extraction of chromosomal DNA was performed using the Sigma GenElute bacterial genomic DNA kit. Digested DNA was purified with MiniElute columns (Qiagen). The Pippin Prep (SAGE science) was used to purify digested fragments of a length comprised between 800 bp and 2 kbp, which corresponds to the region containing the transposon. 400 ng of purified DNA was ligated overnight at 16 °C to adapters with T4 DNA ligase (NEB). Ligated DNA was purified using MiniElute columns (Qiagen). For each library, 3 PCR reactions were performed using 2 μl of ligated DNA as template and a pair of Illumina oligos. The PCR reactions were performed using the following parameters: 98 °C for 30 seconds, 17 cycles of 98 °C 30 seconds, 68 °C 30 seconds, 72 °C 30 seconds and a final step of 72 °C for 7 minutes. PCR products were concentrated with a MiniElute columns (Qiagen) and purified using Pippin Prep (SAGE science).
ENA-FIRST-PUBLIC
2020-07-07T17:22:43Z
ENA-LAST-UPDATE
2020-04-15T21:10:24Z
External Id
SAMEA6765935
INSDC center name
Universite Paris-Saclay, CEA, CNRS
INSDC first public
2020-07-07T17:22:43Z
INSDC last update
2020-04-15T21:10:24Z
INSDC status
public
Submitter Id
E-MTAB-8946:FX126
broker name
ArrayExpress
genotype
EEV29 (N16961 + PbadCre)
growth condition
LB 30C
material type
SC2 reporter
sample name
E-MTAB-8946:FX126
scientific_name
Vibrio cholerae