Figure 1
From: Increasing protein production by directed vector backbone evolution
The five epMEGAWHOP steps to increase protein production by vector backbone mutagenesis. Step I: megaprimer generation, Step II: amplification of the vector backbone under error-prone conditions (0.05 mM Mn2+), Step III: transformation into E. coli DH5α and isolation of plasmids. The expression hosts (E. coli BL21-Gold (DE3) or B. subtilis DB104) were transformed subsequently with the isolated plasmids, Step IV: agar plate pre-screening with selection based on halo formation, and Step V: screening in microtiter plate format to quantify increase in enzyme production.