Cell growth and recombinant expression of alcohol dehydrogenase from Lactobacillus and multifunctional enzyme type 2 (MFE2) from Drosophila in E. coli in shake flask cultures are shown in Figure 3. Induction of 0.4 mM IPTG is indicated with a dashed vertical line. The graph of EnBase cultivation shows that final OD600 of 32 is obtained with expression of ADH in 50 mL shake-flask cultivations.
Remarkably, only cultures with EnBase were able to ensure the maintenance of favorable pH during the extended induction period (up to 24 hours after induction). Stable pH is one reason for the improved protein production clearly visible in Figure 3.
The impact of various aeration conditions is shown in results from different cultivation formats. This is visible in the ADH-producing strain grown in 24-deep-well plates, which reached a final OD600 of 51 compared to OD600 of 32 in shake flasks. Likewise, the MFE2-producing strain reached OD600 of 31 compared to OD600 of 24 in shake flasks.
Additional testing using Ultra Yield Flasks has shown some promising results with newly designed seals that facilitate optimal gas exchange. Increased mixing and oxygenation resulted in new cultivation records when RB 791 was cultivated with EnPresso: OD600 of 78 and cell dry weight of 20 g/L was achieved. This information verifies that proper aeration is important and the combination of EnBase and Ultra Yield Flasks provides the best results.
Controlled cell growth of bacterial cultures at small scale is beneficial for automated high-throughput methods. The constant glucose feed under growth-limiting conditions enables reliable, reproducible cultivation characteristics. A controlled glucose supply provides a longer exponential cell growth phase, which in practice allows a wide time frame for induction.
Traditionally applied cultivation methods using IPTG induction are fixed to an induction time with low cell densities of about OD600 of 0.6. The alternative induction method using lactose as an inducer, called autoinduction, makes the addition of an inducer unnecessary. Unfortunately, the reported expression yield is not adequate in all cases, as the strong induction might lead to a high level of aggregated protein amounts.
The use of EnBase Flo or EnPresso saves both time and labor due to higher volumetric yields. When lower culture volume is needed, it significantly reduces the amount of time and effort required for downstream processing or process optimization. This system could potentially replace standard shake-flask cultivations in the near future.