Figure 5

The recombinogenic capacity of OTG extracts from autoinduced PPY and NEB 5-alpha extracts are equivalent or better than PPY-extracts generated by the original protocol. (a) Plasmid map of pT7-HindIII-ccdB used to assess three-fragment ZeBRα assemblies. Two HindIII and two BsaI sites flank the toxic-placeholder-ccdB, allowing linearization and removal of ccdB. Unique sites are available on either side of ccdB. Chloramphenicol-acetyl-transferase coding gene (CmR), is part of the placeholder cassette and prevents ccdB-loss during plasmid propagation. The hatched region encompasses the fragment removed during cloning. (b) Map of the vector pT7-GFP antisense resulting from the three-fragment test-assembly of the pT7-HindIII-ccdB as recipient for a GFP-ORF and a bacterial promoter containing PCR-fragment, to evaluate the efficacy of the ZeBRα-procedure. Criss-cross lines mark the fusion-sites of the assembled fragments. (c) Comparison of the recombination capacity of extracts prepared with OTG or CelLyticB^TM from manually induced and autoinduced (denoted as “auto” in the column) PPY-cells and NEB 5-alpha. The iVEC/“transformation-cloning” of the respective fragments is shown as last column. (d) Green fluorescent NEB 5-alpha colonies harboring the constitutively GFP-expressing vector pT7-GFP antisense.