Team:Hannover/Design
Design: Our TALebot-vectors and their assembly
In order to generate a TALebot, we used Gibson Assembly to ligate several DNA fragments in a given order. All linkers, tags, the nuclear localization sequence and the repeats were inserted into the iGEM vector as part of PCR amplification primers with overlapping sequences allowing Gibson Assembly to produce the desired TALE.
Designing cyclic TALEs allows a regulation of those proteins, because of topological problems. A TALE is always winding itself around the DNA to bind. If the protein is cyclic, this is no longer possible and the TALE-bond is inhibited. This could also be used for appliances concerning drug delivery. If the cyclic bond is irreversible and a protease can cut the protein, the TALE regains full transcriptional activity (Lonzarić, 2016). To prove this statement, we inserted a TEV cleavage site from the tobacco each virus into the vector which enables induced linearization of the protein after expression with ProTEV Plus protease (Promega).
A Strep Tag with two linker sequences by gBlocks was added to the vector to allow a purification of the expressed protein. In this way, we were able to perform anon-column purification with the Strep-tag system developed by IBA.
Two of our vectors also include an eGFP. Thir green fluorescent protein can be used to detect samples under blue or UV light due to emission of green light. eGFP is an enhanced version of GFP with a higher intensity from Aequorea Victoria. During our experiments, we used eGFP to detect TALEs on the chip spotted with specific DNA.
First, we used BL21 (DE3) cells with a T7 IPTG-inducable promoter to express our protein. But we came across several problems. Our expressed TALE was not circular and a TEV cleavage lead to no results. This is why we did some research and tried purification with DTT as well as expressing our protein in Origami B (DE3) cells. This is a mutation of the original Origami strain enabling the induction of expression with IPTG. In addition and a very important fact for us, Origami strains have mutations in glutathione reductase (gor) and thioredoxin reductase (trxB) which allow disulfide bonding formation in the cytoplasma. DTT and the expression in Origami cells allowed the cyclization of TALEs by stabilizing the peptide bond by disulfide bonds.
