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Fig. 4 | Microbial Cell Factories

Fig. 4

From: A synbio approach for selection of highly expressed gene variants in Gram-positive bacteria

Fig. 4

Production optimization of the industrially relevant proteins tyrosine ammonia lyase (TAL) and sialidase (SIA). a A His-tagged sialidase-encoding sequence was translationally coupled to the chloramphenicol resistance gene. A TIR library was constructed, transformed into B. subtilis and grown with different chloramphenicol concentrations on agar plates. Colony forming units (CFUs) were counted for all concentrations for the library (TIRlib) and the original, non-randomized clone (TIROrig) as control. b A Strep-tagged tal-encoding sequence was translationally coupled to the chloramphenicol resistance gene. A TIR library was constructed, transformed into L. lactis and grown with different chloramphenicol concentrations on agar plates. CFUs were counted for all concentrations for the library (TIRlib) and the original, non-randomized clone (TIRorig) as control. c The sialidase production level in the culture supernatant of the best performing clone (TIRopt) was analyzed by Western blot, using an antibody against a His-tag (left panel). The increase in production was analyzed by densitometry and plotted as the relative protein production compared to the original clone (right panel). d TAL production level of the best performing clone (TIRopt) was analyzed by Western blotting using an antibody against Strep-tag (left panel). The increase in production was analyzed by densitometry and plotted as the relative protein production compared to the original clone (right panel). e Sialidase (TIRopt) was purified via a Ni2+-NTA column and purified product concentration was estimated using a BCA assay (left panel). Product per cell mass and per culture volume was calculated (right panel). The original construct (TIRorig) was used for comparison

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