Coding

Part:BBa_K3944008

Designed by: Olof Dahlman   Group: iGEM21_Chalmers-Gothenburg   (2021-09-20)


ScCUP1 + TesBT

Part containing copper-induced promoter and thioesterase TesBT with specificity for fatty acid chain lengths C4-C8, terminating fatty synthesis and yielding a specific fatty acid profile. This part is ready to use in yeast.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

Thioesterase TesBT originates from the bacterial species Bacteroides thetaiotaomicron. It is a thioesterase enzyme responsible for cleaving the Acp moiety from the growing fatty acid chain, terminating its synthesis and determining the fatty acid profile of the bacteria. The copper-induced promoter CUP1 is native to yeast and is activated when the copper-binding protein ACE1 (2) binds to it following ligand binding to copper. The yeast then expresses metallothioneins which bind and sequester potentially harmful metall ions, such as CU2+. A small amount of copper is necessary to the growth and survival of the yeast however, and as such most growth media contain it and this system tends to have leaky expression.

Since the system is native to yeast, any gene of interest need only be inserted behind the CUP1 promoter and transformed or integrated into the yeast to build a functional, if leaky, inducible expression system. Thioesterase induction the native yeast FAS system needs to be replaced prior however, as the native FAS1 system is a dimer of two large protein strands forming a large complex, which includes thioesterase, leading to competition between native and induced thioesterase without any ability for detailed control.

Experimental design

Copying the CUP1 promoter and TesBT sequences from template via PCR enables the insertion of overlapping overhangs at the primer design stage, which enables us to make use of quick and efficient one-pot Gibson assembly reaction to insert the promoter and thioesterase in the backbone. The created plasmid is then ready to use.

Method

Templates of CUP1 and TesBT ordered from IDT 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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