System for inducible transcription of thioesterase TesA as to regulate fatty acid chain length synthesised in the cell, dependent on level of added tetracycline family antibiotic. Consists of 7 TetO tetracycline recognition sequence repeats followed by S.Cerevisiae phosphate system-derived promoter PHO5 and the thioesterase TesA. Requires TetA tetracycline-sensing protein chimera present in the nucleus to function, see here. For an overview of the whole system, please visit our wiki.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 623
- 1000Illegal BsaI site found at 5
Usage and Biology
Thioesterase TesA originates from the well-known bacterial organism E.coli (2). 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. TesA targets fatty acid chains with a length between 12 to 16 carbons, favouring 16.
The tetracycline transcriptional system originates from bacterial antibiotic resistance genes, reacting to the addition of members from the tetracycline family of antibiotics. In bacterial origins, it triggers transcription of genes downstream of TetO sequence repeats + a promoter of choice, conferring antibiotic resistance to the cell. By moving this system to other organisms, a reliable if somewhat leaky way of expressing desired genes can be obtained. Seven TetO repeats has been found to be optimal (1) and by placing these before a promoter of choice, such as ScPHO5, the downstream gene will be transcribed. As the system requires the introduction of the tetracycline-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.
The seven TetO repeats were obtained on a plasmid with the ScPHO5, optimized for use in S.Cerevisia. By designing primer pairs that leaves the TetO/ScPHO5 and TesA sequence with overlaps to one another, the TetA sensing protein and the p415TEF 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, and designed as such that the promoter/gene region is inserted in the reverse direction to avoid potential transcribing/overlap errors with the TetA sensing protein.
Templates of TetO7/ScPHO5 and TesA 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.