Difference between revisions of "Team:UCopenhagen/Gene Construct Design"
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Revision as of 13:35, 17 October 2018
Design of Gene Construct
1. Expression plasmid without chaperone genes
This plasmid consists of signal-tagged reporter gene controlled by ara operon, which can be activated by adding arabinose to the culture media. The T3SS signal is N-terminal 20 amino acid sequences from E. coli effector proteins Map. Those signals are linked with the reporter protein by a glycine-serine linker peptide. 6X-His tag is commonly used as a purification tag, but in this case, it can be used as an epitope tag for western blot.
How to make it?
2. Expression plasmid with chaperone genes
This plasmid consists of signal-tagged reporter gene controlled by ara operon, which can be activated by adding arabinose to the culture media. It also express CesT and CesF effector chaperone operon under a constitutive promoter pCat. Those chaperones assist natural effector protein transport. It has been excluded from the modified LEE region in SIEC strain, but the author put it in the substrate plasmid instead.
How to make it?
a. Construct pBAD vector with CesF/CesT chaperone.
b. Insert reporter genes with Map20 signal into pBAD-Chaperone plasmid
3. EspD Translocon expression plasmid
This plasmid encodes 6His-tagged EspD translocon under pTac, which can be induced by IPTG. The EspD translocon protein are self-assembled hydrophobic membrane protein that sufficient for membrane penetration and pore formation. This translocon protein are normally secreted along with EspB hydrophilic translocon to penetrate host cell membrane and serve as a docking site for the injectisome. This 6His-tagged EspD can be purified by Ni-NTA column or TALON column. It might be used to pre-assemble a translocon on a target membrane to induce injectisome docking on an artificial membrane.
How to make it?
Biobrick plasmid to be submitted to iGEM
1. Signal sequence biobricks
2. 6x His-tagged CesT/CesF Chaperone biobrick
3. 6x His-tagged EspD translocon biobrick