Journal: Microbial Cell Factories
Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides
Figure Lengend Snippet: Cloning efficiencies for the Golden Gate assembly of different E. coli expression plasmids. Each dot represents one cloning procedure with an individual expression plasmid. There were no significant differences in the cloning efficiencies of the different promoters ( a ), fusion partners ( b ) or proteins ( c ). d Shows the cloning efficiencies for an expression plasmid where two consecutive plasmid features were left out, here the fusion partner and the protease cleavage site. In this case, Golden Gate cloning using two short dummy fragments was compared to a workaround strategy in which one of the neighboring plasmid feature sequences (here for the His 6 tag) carries a Bsa I overhang directly complementary to the plasmid feature “protein of interest” so that no short dummy fragments are needed. As a comparison, the cloning efficiencies for the assembly of the other 18 expression plasmids, which carry all six classes of plasmid features, are also shown
Article Snippet: Each Golden Gate cloning reaction required 100 ng of the acceptor plasmid (pJ915-lacZ), 100 ng of each donor plasmid, 2.5 U Bsa I/ Bsa I-HF and 300 U T4 DNA ligase (NEB, 2000 U/µL) in a reaction mixture of 15 µL in 1× T4 DNA ligation buffer (NEB).
Techniques: Clone Assay, Expressing, Plasmid Preparation