Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Miranda Halle
Date created: 2016-07-18 11:00:00
Date modified: 2016-07-25 02:44:12
Cassette for the constitutive production of GAL4-KaiCp and LexA-SasA, linked by a self-cleaving P2A
| Types | DnaRegion |
| Roles | engineered_region Composite |
| Sequences | BBa_K2176001_sequence (Version 1) |
Description
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.
Notes
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.
Source
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.
| Sequence Annotation | Location | Component / Role(s) |
| BioBrick Prefix BioBrick Suffix TEF1 Promoter ADH1 Terminator SV40 Nuclear Localization Signal Gal4 Activator Domain Linker Domain Phosphorylated KaiC Protein P2A Self-Cleaving Linker SV40 Nuclear Localization Signal LexA Protein Linker Domain SasA Protein DNA Binding Domain of LexA Phosphomimetic Variable | 6,27 4465,4485 40,438 4278,4464 458,478 479,817 818,826 827,2383 2384,2449 2450,2470 2471,3076 3077,3106 3107,4270 2471,2722 2042,2047 | feature/BioBrick engineered_region engineered_region feature/BioBrick feature/promoter promoter sequence_feature feature/misc sequence_feature feature/misc non_covalent_binding_site feature/binding feature/misc sequence_feature CDS feature/protein sequence_feature feature/misc feature/misc sequence_feature CDS feature/protein feature/misc sequence_feature CDS feature/protein non_covalent_binding_site feature/binding feature/misc sequence_feature |