BBa_K2176001 1 BBa_K2176001 Cassette for the constitutive production of GAL4-KaiCp and LexA-SasA, linked by a self-cleaving P2A 2016-07-18T11:00:00Z 2016-07-25T02:44:12Z The promoter and terminator in this cassette were amplified from the yeast genome, using primers that added extra overhangs with novel restriction enzyme cut sites on either end: the BioBrick prefix (for the promoter) or suffix (for the terminator) on one side, and an overhang with a cut site for ligating the part to the protein coding sequence on the other side. The protein coding domain was synthesized in G-blocks and constructed via Gibson assembly. None of these components came from their own BioBricks, which is why we've entered the composite as a single "Basic" part. This part is a composite of four parts: 1) a (constitutive) yeast TEF1 promoter 2) a protein coding sequence for a fusion of the GAL4 activator domain and phosphorylated KaiC 3) a protein coding sequence for a fusion of LexA and SasA 4) and a yeast ADH1 terminator The two protein coding sequences are linked by a P2A linker domain, which is self-cleaving. This part works in tandem with a GFP reporter construct we designed (BBa_K2176000) that is regulated by a LexA operator. The LexA protein from this part (that is, BBa_K2176001) binds to the LexA operator, carrying with it the SasA protein to which it is fused. SasA binds phosphorylated KaiC (KaiCp), which is made by this cassette in a 1:1 ratio with the LexA-SasA fusion. This KaiCp is fused with a GAL4 activator domain, which recruits RNA polymerase. As a result, an organism expressing both this cassette and the GFP reporter BBa_K2176000 will be constitutively expressing GFP. false false _2647_ 19613 19613 9 false We chose to express both fusion proteins in one cassette, joined by a self-cleaving linker, in order to obtain equal stoichiometric amounts in the cell. In order to join the promoter and terminator to the protein coding sequence, we used two restriction enzymes, BamHI and BglII, which produce the same sticky ends when cut but have different recognition sites due to differences in the base pairs flanking the sticky ends. The promoter was cut with BamHI, while the side of the protein coding sequence it was to be joined to was cut with BglII. These two were then able to ligate together, which produced a novel 6bp sequence that is half-BamHI site, half-BglII site, and as a result is a recognizable cut site to neither. The same procedure was used to join the protein coding sequence and the terminator. false Miranda Halle annotation2481344 1 P2A Self-Cleaving Linker range2481344 1 2384 2449 annotation2481026 1 BioBrick Suffix range2481026 1 4465 4485 annotation2481346 1 LexA Protein range2481346 1 2471 3076 annotation2481345 1 SV40 Nuclear Localization Signal range2481345 1 2450 2470 annotation2481027 1 TEF1 Promoter range2481027 1 40 438 annotation2481340 1 SV40 Nuclear Localization Signal range2481340 1 458 478 annotation2481025 1 BioBrick Prefix range2481025 1 6 27 annotation2481343 1 Phosphorylated KaiC Protein range2481343 1 827 2383 annotation2481341 1 Gal4 Activator Domain range2481341 1 479 817 annotation2481349 1 DNA Binding Domain of LexA range2481349 1 2471 2722 annotation2481028 1 ADH1 Terminator range2481028 1 4278 4464 annotation2481342 1 Linker Domain range2481342 1 818 826 annotation2481348 1 SasA Protein range2481348 1 3107 4270 annotation2481347 1 Linker Domain range2481347 1 3077 3106 annotation2481350 1 Phosphomimetic Variable range2481350 1 2042 2047 BBa_K2176001_sequence 1 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igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 James Alastair McLaughlin Chris J. Myers 2017-03-06T15:00:00.000Z