pIBAINS

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	The constructed plasmid pIBAINS was used to transform the E. coli 20 strain obtained in our Institute. We used IBA's E. coli 20 strain and IBA's vector because ownership of a patented strain allows the invention to be commercialized.

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The constructed plasmid pIBAINS was used to transform the E. coli 20 strain obtained in our Institute. We used IBA's E. coli 20 strain and IBA's vector because ownership of a patented strain allows the invention to be commercialized.

Publication protocol

"The constructed plasmid pIBAINS was used to transform the E. coli 20 strain obtained in our Institute. We used IBA's E. coli 20 strain and IBA's vector because ownership of a patented strain allows the invention to be commercialized.
Bacterial cells for culture came from glycerol stock stored at −80 °C. E. coli 20 cells, harboring plasmid pIBAINS with the human insulin gene, were grown for 18 h at 30 °C in shaking flasks containing 50 mL GMS medium [11] supplemented with tetracycline (100 μg/mL), proline (600 μg/mL) and thiamine (2 μg/mL) until the optical density at 600 nm was about 0.5–1.0. The flasks were used as an inoculum for a Bioflo Celligen 310 7.5 L fermenter (starting GMS medium volume 4 L). In the fermenter antibiotics were not used. Cells were grown for 15–16 h at 37 °C. During the growth stage of the culture, 40% glucose and 12 mg/mL proline were exponentially added as the carbon source (to maintain glucose concentration range 70–120 mg/dL). The pH was controlled during the entire run by addition of 16% NH4OH. When OD 600 nm reached about 25–35, the glucose feeding was limited until its concentration in the medium was reduced to 0 g/dL. Then, the glucose feeding was connected to a pump with acid (pH-stat control using cascade). Glucose was automatically added by the controller whenever the pH exceeded the set point (dead band was 0.01). The glucose level was maintained by the pH-stat in the concentration range of 30–50 mg/dL. After induction, the culture was grown for 4–5 h until OD 600 nm was about 50–60, reaching the stationary phase of growth."

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