Part:BBa_K3944035
TEF1p-Estra-ScADH1t
Fusion protein consisting of protein zif268 which binds to repeats of the Z3BS recognition sequence, estradiol-recognizing domain ER LDB which triggers conformational change and activity upon binding to estradiol, and viral transcription protein VP16, transcribing genes downstream fusion protein upon binding to DNA sequence. The whole complex will trigger transcription of genes downstream of the Z3BS repeats upon addition of the induction agent estradiol in a dose-dependent manner (1). This protein is required for the estradiol-based induction system to function, as it will not register estradiol induction signals without it. Please visit our wikito learn more.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 580
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 161
Aim
In our project we aim to create a yeast cell where the fatty acid chain length produce can be controlled by three separate induction systems expressing different thioesterases, which will influence the fatty acid profile produced by the cell. In this effort, we needed to introduce this protein, short-hand called Estra or Est, in order for the estradiol induction system to function (2). Without this protein, the Z3BS/ScGAL1 recognition repeat/promoter sequence will not be recognised by anything and there will be no transcription and expression of our desired gene.
Usage and Biology
In the estradiole-based induction system, gene expression is induced by the mammalian hormone β-estradiol. Similar to TET, several parts are required in order to make this system function properly. An estradiol ligand recognizing domain (ER), that alters conformation upon binding to estradiol, has been fused to a human zinc finger protein native to neurons (Zif268). Zif268 recognizes a short DNA sequence known as Z3BS (1). ER-Zif268 is further fused with VP16 to ensure strong activation, and multiple repeats of Z3BS are used to increasing binding efficiency.
Experimental design
The variant of this system we decided to use for our project is an improvement of the original system more optimized for induction in yeast. In the original system, ER was fused with the yeast GAL4 activation domain, making it bind to the endogenous GAL promoters and thereby unwanted influenced the cells metabolism. As other GAL4 regulators could also bind to the promoter, this system could not be used in media containing galactose, unless GAL4 was knocked out. By substituting the GAL4 with Zif268, the variant system allows tightly regulated and growth condition-independent transcription (3). We then further designed primers for the cloning of Estra template so that the overhangs generated could be used for one-pot Gibson assembly to efficiently assemble the p416TEF plasmid backbone with Estra, the strong constitutive promoter TEF1 and the Z3BS/ScGAL1 expression cassette with our desired gene, incorporate so that they are read in forward and reverse directions, minimizing unwanted overlaps or misreads of one sequence over the other.
Methodology
The template for the Estra fusion protein, TEF1 promoter, p416TEF-GFP backbone and Z3BS/ScGAL1 was provided by systems biology department at Chalmers technical highschool. Sequence templates were cloned using Phusion polymerase along, and products were purified using ThermoFischer gel purification protocol and kit. The purified fragments were assembled with either cloned p416-GFP to generate GFP-containing benchmarking plasmid, or with digested linearized p416TEF plasmid backbone, to produce two plasmids bearing Estra protein and the system utilizing the protein to express our desired genes. The produced plasmids were transformed into competent E.coli cell strain DH5-alpha, grown overnight on plate, inoculate overnight again, followed by extraction following protocol using plasmid miniprep kit from ThermFischer scientific. Purified plasmids were then transformed into yeast as required.
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