Figure 6

Rendering the CcdB in the placeholder non-toxic increases the number of GFP^– background colonies markedly, showing that ccdB is an essential element if working with non-gel purified vector. (a) The pT7-HindIII-dead-ccdB differs by a four base-pair deletion in the ccdB-coding sequence from it’s predecessor pT7-HindIII-ccdB. The dotted lines encompass the region removed during cloning. (b) Sequence alignment of the region encompassing the small deletion in the ccdB-ORF, in pT7-HindIII-dead-ccdB compared to the region in pT7-HindIII-ccdB and the resulting frameshift rendering the ΔCcdB non-toxic for E. coli NEB 5-alpha. The numbers denote bases in the vector. (c) The three-fragment assembly as shown in Fig. 6c uses the pT7-HindIII-dead-ccdB vector and OTG-derived PPY-extract to assemble pT7-GFP antisense analogous to the assemblies shown in Fig. 5. The percentage of GFP⁺ colonies drops from nearly 100% for pT7-HindIII-ccdB to 57% for pT7-HindIII-dead-ccdB. (d) Image of the mixture of GFP⁺ and GFP⁻ resulting from the assembly (100 µl outgrowth medium spread). The red arrow points at a cluster of GFP⁻ colonies representing un-digested vector that would have to be screened for in a non-model assembly.