title: Illumina MiSeq sequencing; Predictive design of sigma factor-specific promoters
design: Predictive design of sigma factor-specific promoters
library_name: Sample 31_s
library_construction_protocol: Plasmid libraries were transformed by electroporation into the E. coli K12 MG1655. All strains were cultured overnight (12 h) at 30°C in 5 ml selective 853 medium. Cultures were then resuspended and diluted in filtered phosphate-buffered saline for cell sorting. A BD Influx Cell Sorter (FACS core facility, cmpg, Leuven), calibrated with Rainbow Calibration Particles (8 peaks, 3.0 – 3.4 µm)(BD), was used for cell sorting to split the libraries into separate bins, according to the different promoter TIF levels. Constitutive sfGFP expression was taken into account for bin selection to exclude artifacts. Sorted cells were collected in collection tubes with 1 mL 853 medium supplemented with kanamycin. Subsequently, sorted cells were added to 5 mL fresh 853 medium supplemented with kanamycin in 50 mL tubes and incubated overnight at 30 °C while shaking.  Plasmid DNA was isolated for all individual bins with a Qiagen Plasmid Mini Kit (Qiagen, Venlo, The Netherlands) to prepare for DNA sequencing. Libraries were constructed using standard molecular cloning protocols. Randomized DNA sequences were included in polymerase chain reaction (PCR) primers. CPEC is used subsequently to construct a plasmid. The cloning vector (pLibrary) is based on the pSC101 plasmid and additionally contains a constitutively expressed sfGFP gene. The assembled plasmids were transformed in electrocompetent Top10 cells and subsequently grown overnight in 10 ml selective LB medium at 37°C. Plasmid DNA was extracted for all individual libraries with a Qiagen Plasmid Mini Kit.