Coding

Part:BBa_K3944021

Designed by: Olof Dahlman   Group: iGEM21_Chalmers-Gothenburg   (2021-09-30)


TEF1-TetA-ScPGK1

Full sequence with promoter and terminator for the constitutive production of protein chimera TetA, binding to TetO7 + ScPHO5 recognition sequence + promoter upon addition of induction agent tetracycline or similar compounds. Required for the function of the TetON system, this sequence is ready to use in yeast cells.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 760
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1418
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1331
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 161

Aims

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 TetA, in order for the tetracycline TetON induction system to function (1). Without this protein, the TetO7/ScPHO5 promoter sequence will not be recognised by anything and there will be no transcription and expression of our desired gene.

Usage and Biology

The tetracycline induction system originates from bacterial tetracycline antibiotic resistance, and has since been optimized for use as an induction system in eukaryotic organisms such as yeast (1). In the tetracycline-based induction system (TET), gene expression is induced by the presence, or absence, of members of the tetracyline family of antibiotics. There are two commonly used variants of this system, TetON and TetOFF. The TetON system is based on a reverse tetracycline-controlled transactivator (rtTA) composed of the TetR repressor binding protein from the tetracycline resistance operon of E. coli transposon Tn10. In the original version of the system, TetR is fused to the strong transactivating domain of VP16 (alternatively referred to as vmw65 or α-TIF in the literature) from Herpes simplex virus (2). TetON is a variation of TetOFF in which four amino acids have been modified in the TetR DNA binding moiety which alters its binding characteristics to the promotors. The difference between the two systems is not whether the transactivator turns a gene on or off (as both proteins activate expression). Rather, TetON activates expression in the presence of tetracycline, while and TetOFF activates expression in the absence of tetracyline. The recognition site is made up of Tet operator (TetO) sequences derived from the human cytomegalovirus immediate-early promoter and multiple TetO repeats can be placed in sequence to increase induction efficiency.


Experimental design

In our project we utilize a sequence with VP16 replaced with yeast GAL4 activation domain (3), and rtTA is linked to a nuclear localization signal sequence (4), since the system is native to prokaryotic bacterial cells and thus lack nuclear localization on its own, which is required for function in yeast. We also utilized seven TetO sequence repeats, which has been shown to give the highest expression efficiency, along with a promoter sequence that activates the transcriptional domain, GAL4AD. We designed our experiment as such that by cloning the required TetA, promoter TEF1 to strongly constitutively express TetA once in the cell (5), recognition site/promoter and either TesA or mTagBFP2, we could insert overhangs during the primer design stage such that Gibson assembly could efficiently assemble the TetA protein and requisite expression system on the same plasmid, with the TetA protein transcribed in the reverse direction to avoid undesired transcriptional overlaps or other errors.

Methodology

Main TetA sequence was provided as a template by systems biology department at Chalmers technical highschool. Sequence template was cloned using Phusion polymerase along with TEF1 promoter, and products were purified using ThermoFischer gel purification protocol and kit. The purified fragments were assembled with plasmid p415TEF linearized backbone along with recognition site/promoter and either TesA thioesterase insert for practical use or mTagBFP2 insert for benchmarking of the induction system, to produce two plasmids bearing TetA 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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