Team:USP-UNESP-Brazil/Plasmid Plug n Play/Background

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The Plug and Play project is base on the Cre-lox recombination mechanism, which occurs between the Cre Recombinase enzyme and the loxP sites.

Cre Recombinase (38kDa) is a tyrosine recombinase from the P1 Bacteriophage (1). It is known to play important roles in this organism’s life cycle, such as cyclization of the linear genome when the phage infects the bacteria and resolution of dimeric chromosomes that form after DNA replication (7). The enzyme uses a topoisomerase I like mechanism to carry out site-specific recombination events between two DNA recognition sites (called loxP sites). Each loxP recognition site has 34pb, which consists of two 13bp palindromic sequences that flank an 8bp spacer region (ATAACTTCGTATA–GCATACAT–TATACGAAGTTAT).

The recombination result is dependent to location and relative orientation of the loxP sites, which can be in cis (same DNA strand) or trans (different DNA strands). The spacer sequence (8bp) found in each loxP site is also important for the outcome of the recombination process, it gives the orientation of one site in correlation to the other when both are in cis position. When DNA is found between two loxP sites oriented in the same direction this DNA would be excised as a circular loop, when these two loxP sites are in opposite directions the DNA between them will be inverted (5) (Fig. 1). The enzyme requires no additional cofactors (such as ATP) or accessory proteins for its function (1).

Concerning the molecular mechanism of recombination, a single recombinase molecule binds to each palindromic half of a loxP site, then the recombinase molecules form a tetramer, thus bringing two loxP sites together (8). The recombination occurs within the spacer area of the loxP sites. The post-recombination loxP sites are formed from the two complementary halves of the pre-recombination sites (Fig. 1). This mechanism was further improved for science research purposes, and nowadays, lots of tools have been made based on it. The best-known example is the Nobel Prize winner experiment of knockout on rats using the Cre-lox recombination (4).

Now we will talk about DNA integration or insertion. It is another way of working using Cre-lox recombination between two different DNA fragments. It has already been extensively explored and is the key mechanism of our experiment. Using two DNA fragments, each one with one loxP site, is possible to fuse both into one larger fragment.

However, insertion of a circular DNA carrying a loxP into a loxP site on a chromosome (integrative recombination) is quite inefficient because unimolecular reactions are kinetically favored over bimolecular reactions, causing that the inserted DNA will often be excised (6) (Fig. 2a). To tackle this problem we have used loxP mutant sites (2). These sites help to maintain the inserted DNA in the chromosome (Fig. 2b).In our project we used one loxP site and two loxP mutant sites (lox71 and lox66) (Fig. 2c).