Coding

Part:BBa_K3944033

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


ScGAL1 + Z3BS-FatB

Inducible short recognition sequence repeats/promoter region which induce downstream transcription of thioesterase enzyme FatB upon addition of estradiol, depending on level of induction agent. Sequence is optimized for use in S.Cerevisiae, but requires the presence of estradiol-sensing protein for it to be induced upon estradiol addition, see here. For an overview of the whole project, please visit our wiki.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1491
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 169
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 915
    Illegal BsaI.rc site found at 1285
    Illegal BsaI.rc site found at 1758
    Illegal SapI site found at 734


Usage and Biology

Thioesterase FatB originates from the plant Ricinis Communis (1). It is an enzyme responsible for cleaving the Acp moiety from a growing fatty acid chain, terminating its elongation and determining the overall fatty acid profile of the cell. FatB targets fatty acid chains with a length between 16 to 18 carbons. The estradiol transcriptional system originates from human estradiol hormone signalling pathways, with the estradiol ligand binding domain forming a key signal transduction protein in human hormone signalling (2). Similarly, the zif268 zinc finger domain, which binds to the short recognition sequence Z3BS upon being activated, also originates from the human body, being a key component in the regulation and transcription of human neuronal cells (3). By moving this system to other organisms, a reliable way expressing desired genes can be obtained, since both estradiol and zif268 are tightly regulated in the human body. By placing Z3BS repeats before the GAl1 promoter from S.Cerevisiae, a strong induction can be attained that is tightly regulated by the addition of estradiol. As the system requires the introduction of the estradiol-sensing protein, building a single plasmid with both the promoter/gene system and constitutively expressed sensing protein, a single-transform induction system can be created, making use much easier.


Experimental design

The seven Z3BS repeats were obtained on a plasmid with the ScGAL1 as a single unit, optimized for use in S.Cerevisiae. By designing primer pairs that leaves the Z3BS/ScGAL1 and FatB sequences with overlaps to one another, the Estradiol-sensing protein and the p416TEF plasmid backbone, the plasmid can be assembled in a one-pot Gibson assembly reaction, which has been shown to work with up to 12 insert fragments, designed as such that the promoter/gene region is inserted in the reverse direction to avoid potential transcribing/overlap errors with the estradiol-sensing protein.


Methodology

Templates of Z3BS/ScGAL1 and FatB ordered from IDT or provided by our department were mixed with the corresponding forward/reverse primer pair and PCR reaction performed with Phusion polymerase according to protocol. Backbone-containing E.coli cells were grown overnight and then harvested by following protocol for ThermoFischer plasmid miniprep kit, and subsequently cleaved with restriction enzyme to linearize the backbone. The created insert fragments and cleaved backbone were purified using gel purification and ThermoFischer gel purification kit, followed by assembly using the Gibson assembly method according to protocol. The assembled plasmid was transformed into competent E.coli of strain DH5-alpha, grown overnight, inoculated to be grown overnight again and then harvested with miniprep. The harvested plasmids were sequenced, but due to time constraints, the finished plasmid was not transformed and tested for fatty acid production profile.



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