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. 2015 May 8;43(13):e88. doi: 10.1093/nar/gkv464

Figure 3.

Figure 3.

Assembly and functional testing of transcription units. (A) Schematic representation of the ‘One-POT’ assembly of transcription units. The standard parts (promoters, ORFs, terminators) and the ‘POT’ accept vector were mixed together with buffers and enzymes to assembly the transcription units in one tube. The red colonies are the residual intact acceptor vectors and the white colonies contain the correctly assembled transcription units. (B) Restriction enzyme digestion to confirm the assembled transcription units. Five or six white colonies were randomly picked for three different transcription units, and all of them showed the correct insert and vector at the expected size. TU1 is pTDH3-crtE-tTEF1 into POT2; TU2 is pADH1-crtI-tADH1 into POT4, and TU3 is pTEF2-crtYB-tTEF2 into POT5. (C) Western blotting to detect the expression of assembled TUs. Three human ORFs were tagged by HA or Flag epitope and assembled into the POT vectors under the control of TEF2 or GALs promoters. Two clones were randomly isolated after the plasmids were transformed into yeast. The expression of these proteins was detected using antibodies against HA or Flag tag. (D) Western blotting to detect the expression of a human complex integrated into the yeast genome. All five components are detectable using antibodies against the epitopes although the level of expression is not uniform.