ERC000011
ENA-CHECKLIST
2022-07-11
ENA-FIRST-PUBLIC
2022-07-11
ENA-LAST-UPDATE
SAMEA8647904
External Id
CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology
INSDC center alias
CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology
INSDC center name
2022-07-11T00:19:23Z
INSDC first public
2022-07-11T00:19:23Z
INSDC last update
public
INSDC status
ERS6332465
SRA accession
E-MTAB-10380:Cm_A_10_3
Submitter Id
ArrayExpress
broker name
Protocols: M. pneumoniae cells were grown in two T75 cm2 flasks and recovered in a 2ml electroporation buffer and 80 µl of cells were electroporated with 2 pmol of the different mini-transposon vectors (2 technical replicates per sample and two biological replicates). After electroporation cells were resuspended in a final volume of 1 ml by adding 900 µl of HF. The 2 transformations of each vector were pooled (total volume of 2ml). Five hundred µl of cells were cultured in 20ml of medium in a T75 flask with different concentrations of chloramphenicol (0.5, 1, 2 and 15 µg/ml) for 4 days at 37ºC in 5% CO2. After one day of incubation each flask was resuspended in 1.5 ml of medium and cells were seeded in 150 ml of medium in a T300 flask. After 48 days of growth at 37ºC in 5% CO2 DNA of samples treated with 0.5, 1, 2 µg/ml of chloramphenicol was extracted. The samples of 15 µg/ml were processed after 72 additional hours. This experiment was repeated twice and DNA samples were sequenced independently. M. pneumoniae M129 strain was grown in 75 cm2 tissue culture flasks with 50 mL of modified Hayflick medium (HF) at 37ºC and was transformed as previously described 45. To select M. pneumoniae transformant cells, plates were supplemented with 20 µg/ml chloramphenicol or 0.02 µg/ml of erythromycin. Transformed cells were also grown in liquid cultures and testing different concentrations of antibiotics. First, M. pneumoniae M129 was grown in a 96 well plate format with 200 µl of HF and 5 µl of transformed cells. For chloramphenicol the tested concentrations were 0, 0.5, 1, 2, 5, 10, 15 and 20 µg/ml. In the case of erythromycin, the tested concentrations were 0,002 and 0.02 µg/ml. Concentrations of 0.5, 1, 2, 5, 10, 15 µg/ml for chloramphenicol libraries and 0.02 µg/ml for erythromycin libraries were selected for sequencing. To study the proteome of M. pneumoniae, transformed cells were grown in T75 flasks with different antibiotic concentrations (0.5, 1, 2 and 15 µg/ml of chloramphenicol) to cover from low to highly expressed proteins. After 24 h cells were passed to a T300 cm2 flask, cultures of cells grown with 0.5, 1, 2 and 15 µg/ml of antibiotic were confluent after 48h and cultures of cells grown in 15 µg/ml required three additional days. Genomic DNAs of M. pneumoniae M129 were isolated with the Illustrabacteria genomic Kit (GE). The purification of PCR products and digested fragments from agarose gels were achieved using the QIAquick Gel Extraction Kit (Quiagen). Plasmid DNA was obtained by using the QIAprep Spin Miniprep Kit (Quiagen). Between 10 and 500 ng genomic DNA were fragmented to 200-300 bp using a Covaris S2 instrument. End repair and adaptor ligation was performed using the NEBNext Ultra DNA Library Prep kit for Illumina according to manufacturer's instructions with the exception that the adaptor used contained only the read 1 adaptor sequence and not the standard Illumina Y-shaped adaptor containing read 1 and read 2 adaptor sequences. The adaptor ligated dnA was amplified with NEBNext Q5 Hot Start HiFi PCR Master Mix in a 50 µL reaction with primers R1 PA primer and Tn select PA primer (0.2 µM final concentration) using the following PCR program: 98 °C, 30 seconds; 8 cycles of 98 °C, 10 seconds and 65 °C 25 seconds; followed by a final extension of 5 minutes at 65 °C. An estimation of PCR cycles required for library amplification was performed by qPCR NEBNext Q5 Hot Start HiFi PCR Master Mix in a 50 µL reaction adding SYBR Green I (10.000x in DMSO Sigma Aldrich) to the PCR reaction at 0.1x final concentration. PCR was performed in a Roche Light Cycler LC480 for 30 cycles using the same conditions as for the first PCR reaction. 1 µL of the first PCR was used as template and Universal PCR Primer (NEB) and R2 TN select nested primer mix were used at 0.2 µM final concentration. The remaining 49 µL of the first PCR were purified using 1.8x volumes of AMPure XP beads (Beckman Coulter) according to the manufacturer's protocol. The purified product was eluted in 48 µL of buffer EB (Qiagen). 15 µL of the purified first PCR product were then used for a second PCR with the number of cycles estimated from the previous qPCR (cycle number close to plateau minus 3 cycles due to the increased amount of template). PCR conditions were the same as in the qPCR using NEBNext Q5 Hot Start HiFi PCR Master Mix in a 50 µL reaction and Universal PCR Primer (NEB) and R2 TN select nested primer mix were used at 0.2 µM final concentration but omitting the SYBR Green I. The second PCR was purified using 1x volume of AMPure XP beads and eluted in 20 µL of buffer EB. To complete adaptor sequences and add sample barcodes, a third PCR was performed with NEBNext Q5 Hot Start HiFi PCR Master Mix in a 50 µL reaction using 19 µL of the purified second PCR as templates. Primers used were Universal PCR Primer and a suitable NEBNext Multiplex primer for Illumina at 0.6 uM final concentration. The PCR program used was: 98 °C, 30 seconds; 4 cycles of 98 °C, 10 seconds and 65 °C 75 seconds; followed by a final extension of 5 minutes at 65 °C. After the third PCR libraries were purified using 1x volume AMPure XP beads and eluted in 20 µL buffer EB.
description
Random mutagenesis with TnP438catIR*
genotype
Mycoplasma pneumoniae M129
http://purl.obolibrary.org/obo/NCBITaxon_272634
organism
Mycoplasma pneumoniae M129
organism
whole organism
organism part
Cm_A_10_3
title