Robustness of gene expression and growth to large-scale gene translocation

Experimental study of the robustness of gene expression and growth of Escherichia coli K-12 MG1655 to large-scale gene translocation.Strains were streaked onto LB agar plates containing tetracycline [3 ug/mL] and incubated at 37 C overnight. Three individual colonies per strain were separately transferred into 2 mL glucose [0.2% w/v]-minimal medium supplemented with tetracycline [3 ug/mL] and pre-cultured at 37 C while shaking (250 rpm) for approximately 24 hours. 100 uL of pre-culture was transferred into 10 mL of glucose [0.2% w/v]-minimal medium supplemented with tetracycline [3 ug/mL]. Two replicate cultures were prepared from each pre-culture. Strains were cultured in 50 mL Falcon tubes at 37 C while shaking (250 rpm) for up to 24 hours. One replicate culture of each pre-culture was sampled after 4 hours, while the other replicate was sampled after 24 hours. At each sampling time point, OD600 and A600 was measured, cultures were placed on ice, spun down (5000 x g, 10 minutes, 4 C), resuspended in 1 mL ice-cold M9 minimal medium without any addition, transferred to a 1.5 mL Eppendorf tube, spun down (5000 x g, 1 minute, 4 C), the supernatant was carefully removed via pipetting, cell pellets were flash frozen in liquid nitrogen, and samples were stored at -70 C. Within each sampling time point samples were processed in parallel. Samples were shipped on dry ice to Genewiz (Germany) for RNA extraction, rRNA depletion, library preparation, and sequencing. Sequencing was performed on an Illumina NovaSeq 2x150 platform. Sequencing results were delivered in fastq format. Approximately 10 million reads were produced for each sample.