Difference between revisions of "Part:BBa K1362453"

Revision as of 14:09, 21 October 2019

TEV protease recognition/cleavage site

This part codes for the amino acid sequence ENLYFQG. This site is recognized by TEV protease (catalytic domain of the Tobacco Etch Virus nuclear inclusion a (NIa) protein), which will cleave the Gln-Gly peptide bond [1]. The final four nucleotides of this sequence are GGGT, which will be the overhang produced wenn a reverse-facing BsaI site (BBa_K1362423, BBa_K1362427, BBa_K1362447) is directly following this part, as in the Sortase A circularization constructs (BBa_K1362202, BBa_K1362203, BBa_K1362204, BBa_K1362205).

• TEV Protease is a recombinant protease derived from Tobacco Etch Virus (TEV) Nla, which is used to excise the affinity tag of the purified fusion protein. TEV protease has strong site specificity and can recognize the seven amino acid sequence of EXXYXQ(G/S) (Glu-Asn-Leu- Tyr-Phe-Gln-Gly). The most common one is ENLYFQG, and its cleavage site is at Between glutamine and glycine or serine.

Experimental Results

• Using the specificity of TEVp, we cleave the recognition sites of ALKBH (Fig.A) and Casp3 (Fig.B) linked by the target sequence, which are active.

• Fig.A: AND logic gate. The reporter was used at a concentration of 150 ng, the fluorescence value was measured at 515 nm, and the ordinate was the normalized fluorescence intensity value. The and logic gate pathways show that when TEVP and TVMVP are present alone, the fluorescence value at 515 nm is very low, and when both are present, the fluorescence value is high, indicating that both proteins are required to function in the pathway.
• Fig.B: OR logic gate. The reporter was used at a concentration of 150 ng, the fluorescence value was measured at 515 nm, and the ordinate was the normalized fluorescence intensity value. The or logic gate pathway shows that when TEVP and TVMVP are present alone, the fluorescence value at 515 nm is high, and when both are present, the fluorescence value is very low, indicating that two proteins are required to function in the pathway alone.
• Fig C: NOR logic gate. The reporter was used at a concentration of 150 ng, and the fluorescence value was measured at 515 nm. The norm gate showed that the ordinate was the normalized fluorescence intensity value. When TEVP and TVMVP are present, the fluorescence value at 515 nm is very low, and when both are absent, the fluorescence value is high, indicating that it is necessary to have two proteins at the same time in the pathway to function.

Usage and Biology

Sequence and Features