Figure 5.
Use of secondary sites to screen variants of a transcription factor gene. (A) The parts used for the construction of the cIts library were: a constitutive promoter, an RBS+N-terminus part, cIts CDS without N or C terminus, and C-terminus + Terminator. The RBS+N-terminus part was PCR-built from a degenerate oligonucleotide variable for the Shine–Dalgarno consensus and for the codon following the start ATG, thus giving the library diversity in translation initiation and half-life of the protein (40). (B) Colonies that were white at 30°C were tested for expression of the mCherry reporter at 37°C. As a control, we analyzed the fluorescence of two colonies from the library that were unrepressed at 30°C (clones G4 and H1), two colonies with a constitutive mCherry gene, and four colonies from an equivalent library assembled with the wild-type (thermostable) cI CDS part.