Cloning and Protein Expression in E. coli

For combined cloning of plasmids and subsequent expression of proteins thereof, AQUA Cloning was performed using 25 μL of BL21 (DE3), or TOP10 E. coli cells as a control, as described above. After transformation, cells were grown overnight in 10 mL LB-medium containing 50 μg/μL spectinomycin at 37°C, 150 r.p.m. The next morning, the overnight culture was used to inoculate 200 mL fresh, antibiotic-containing LB-medium which was induced with 1 mM IPTG when OD₆₀₀ = 0.9 was reached at 37°C, 150 r.p.m. Cells were harvested 6 h post induction by centrifugation at 5,000 g for 5 min, washed with PBS and resuspended in 2 mL PBS before an image was acquired.