Part:BBa_K243000
Protein domain (active) of the restriction endonuclease FokI
This part is used as the active domain of our universal restriction endonuclease. It cut DNA, when it fused with the inactive protein domain of our universal restriction endonuclease(BBa_K243001)and linked with specific oligonucleotides hybridized to DNA.
Introduction
We focused our project on coupling and optimizing the characteristics of a restriction endonuclease with short oligonucleotides to develop a programmable and highly specific enzyme-oligo-complex. As a restriction endonuclease we chose the cleavage domain of the well studied endonuclease FokI from Flavobacterium okeanokoites. Normally FokI acts as a homodimer, each dimer divided in cleavage and restriction domain. Chandrasegaran and Miller have already made experiments to uncouple the cleavage and restriction domains of FokI and created a novel site-specific endonuclease by linking the cleavage domain to zinc finger proteins. For our project we generated two Fok heterodimers (Miller, Nature biotech, 2007) and this part act as the active cutting domain of our universal endonuclease.
Sequence specific nuclease(1968)
The researchers H.O. Smith, K.W. Wilcox, and T.J. Kelley (Johns Hopkins University 1968), were the first persons who isolated and characterized the first restriction nuclease whose functioning depended on a specific DNA nucleotide sequence. This was a big breakthrough for the genetic engineering, it gave the scientists a tool for working with the DNA. Now over forty years later over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially and are routinely used for DNA modification and manipulation in laboratories.
Usage and Biology
The usage of an universal endonuclease could change the daily routine of a scientist, who is working with DNA, because the question where to cut with which enzyme isn't needed anymore. He is free to chose the cutting sequence and can received the part that he wanted. The only thing is he had to plan where to cut and ordered specific oligos.
The idea
The idea is based of the modification of the existing restriction endonuclease FokI. Through modifications in the amino-acids sequence the DNA binding domain was diluted and one cleavage domain was inactivated.
The process for cutting begins when two heterodimeric partners were fused to different anticalins binding different adapter molecules. Thus Fok_i is fused to anticalin on Fluorescein and Fok_a to anticalin on Digoxigenin. These adapter molecules are linked to oligonucleotides mediating the binding of the DNA site of interest. Now the heterodimerization comes into play. If the different Fok_i and Fok_a constructs bind their target oligos and come together, the inactive domain will serve simply as an activator of the active domain, cutting only one strand of the DNA.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 487
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