Journal: Microbial Cell Factories

Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides

doi: 10.1186/s12934-017-0637-5

Figure Lengend Snippet: Golden Gate assembly of the expression plasmid in a single reaction. Six donor plasmids and one acceptor plasmid were combined to form each expression plasmid by directional cloning in a restriction-ligation step using the endonuclease Bsa I and T4 DNA ligase. The overhangs produced by Bsa I are colored

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: Expressing, Plasmid Preparation, Clone Assay, Ligation, Produced

Journal: Microbial Cell Factories

Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides

doi: 10.1186/s12934-017-0637-5

Figure Lengend Snippet: Basic plasmid library for E. coli . The plasmid library consists of 13 donor plasmids, resulting in 27 different expression plasmids. Three promoters (T7/ lac , T5/ lac and araBAD ) were combined with a His 6 tag and either GST or Trx as a fusion partner (or no fusion partner). A thrombin cleavage site was also included in the constructs with a fusion partner. The library includes three AMP genes encoding IMPI, BR021 or the L. sericata antifungal peptide (AFP). The T7 transcriptional terminator was present in all constructs. The chosen Bsa I overhangs are shown above the elements

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: Plasmid Preparation, Expressing, Construct

Journal: Microbial Cell Factories

Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides

doi: 10.1186/s12934-017-0637-5

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