Part:BBa_K1965022
nLuc:AP4:TEVs:P3mS-2A
Introduction
Similar to nLuc:AP4:GS6:TEVs:GS6:P3mS (BBa_K1965021 ), this part consisted of the N-terminus of the split firefly luciferase connected to the AP4 coil, TEV protease substrate and P3mS-2A coil and it goes in combination with P3:cLuc (BBa_K1965005). The parental P3mS sequence is based on P3 coil sequence, which featured substitutions of Ala at the b and c position of the coil heptades with Ser and Gln, to increase its solubility, thereby easing its dissociation from AP4. The coil P3mS-2A sequence was similar to that of P3mS, but was further destabilized by incorporating Ala at the a and d position of the second heptad, which lowered the strenght of the hydrophobic interaction between AP4 and P3mS-2A. This further eased its dissociation from AP4.
Upon activation of TEVp, the linker between AP4 and P3mS-2A, which features the TEVp cleavage site it is cleaved, which enables P3mS to dissociate from AP4. After the dissociation of P3mS-2A, AP4 is free to dimerize with P3:cLuc, which bring the nLuc part into close proximity of cLuc, enabling the split luciferase to regain its catalytic activity 1.
Upon addition of TEV protease the coil P3mS or P3mS-2A, which is now cleaved off from AP4, it is replaced by P3mS:cLuc, thereby promoting the reconstitution of firefly luciferase
Characterization
The construct was used in logic operations, whereby only in the presence of the protease the split luciferase was able to reconstitute and regain its activity (2).
Normalized luciferase activity was compared between samples with and without added TEVp to calculate the fold change of luciferase activity. Out of the four different constructs, the constructs which contained the inhibitory coils P3mS and P3mS-2A worked best, where we observed up to 15 times fold increase with the addition of TEVp.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1723
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 103
Illegal NgoMIV site found at 1447
Illegal NgoMIV site found at 1468
Illegal AgeI site found at 1171 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1538
Illegal SapI site found at 1694
Illegal SapI.rc site found at 1353
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