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·The targeted gene replacement system mainly adopts the principle of homologous gene recombination. We import homologous gene sequenc es to the nuclear genome specific receptor gene loci and replace the original genes, so that we can achieve our purpose of modifying the original genome. In the process of target gene replacement, the construction of vectors is of great importance, which typically includes 2 arms, respectively in homology with the target gene’s homologous sequences on both sides, and 1 selection marker gene which is used to eliminate noise bacteria. In our universal system, we choose GlaA5 and GlaA3 as the homologous arms on either side of the target g ene site and HPH (hygromycin) as selective marker.
· Our system use fluorescent proteins as visual markers for selecting, thus pick out the transformants and distinguish the homologous recombination and non-homologous end joining. And our recombination vector is design for the structure of GlaA5-amilCP-GlaA3-pgPDA(promoter)-cjBlue–linker-HPH-GlaA3, where the homologous gene replacement will occur between homology arms, and the whole cassette will be integrated at the GlaA site, taking replacement of the original gene, so that the blue and purple fluorescence will express at the same time .
· The efficiency of this process is high enough, even achieve 100%. While in the strains where the recombination occurred twice, the colony will only have blue fluorescence, indicating that the selective marker HPH has already been deleted. The purple colonies will be our target transformants. And the fluorescent protein can be used as our tool to make the target gene replacement system visual!
· For the next step, the amilCP is replaced by the adaptor* element, which works as the loader to connect the genes that we want to be expressed to the vector. When the target genes are imported to the host genome’s GlaA specific site successfully, the purple fluorescence will disappear. So that we can distinguish our target transformants!
· Amy
* Adaptor : a segment of DNA containing NotI and XbaI sites. The two sides of itself are two kinds of restriction enzyme cutting sites, prospectively ApaI and HindIII, meanwhile located between two homology arms:GlaA5 and GlaA3 of the plasmid .So, we could achieve the standardization of sites by connected adapoter to GlaA5 and GlaA3.
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