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| M13 Phage Mechanism |
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In our project, the CRISPR system is been transported by phage. So how can phage recognized which DNA it should package and spread? That is accessed by phagemid and helper phage. |
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| Fig.1 |
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Take M13KO7 helper phage and phagemid pBluescript II SK(-) as example, as we use them in our experiment. M13KO7 helper phage has complete coat proteins and a complete genome just like normal M13 phage. But its f1 ori has been inserted by a p15A ori and a kanamycin resistance gene. While in pBluescript, its structure is like a normal plasmid but carries two replication origin: a high efficient PUC ori for itself, and an additional normal f1 ori.
In other phagemid and helper phage pairs, situations are same: helper phage’s replication origin is mutated while phagemid contains an additional replication origin for this helper phage. |
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| Fig.2 |
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When a normal M13 phage infect E.coli with F plasmid(strain JM101 in our experiment), it will use F pilus to put its genome into cytosol. Then this single strand genome will use host’s polymerase to make itself a double strand structure and stay in cytosol like a plasmid.
M13 genome contains major 9 genes, we call each gene’s product as gp1, gp2, etc. When it wants to produce progeny phage, gp2 will recognize f1 ori and make a single strand break on genome. Then gp5 will form dimer structure and start to package this single strand DNA from package signal on f1 ori, this will help stabilize single strand genome in cytosol. After that, progeny phage will be released from host cell and be coated by gp3, gp6, gp7, gp8, gp9. |
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When it comes to M13KO7 helper phage, it still can successfully infect E.coli with F plasmidand make its genome stable in cytosol by host’s polymerase. But because its f1 ori has been mutated, so gp2 and gp5 cannot make a nick and start packaging. This means, helper phage cannot produce progeny phage itself.
However, if this E.coli has been transferred with a pBluescript II SK(-), gp2 will recognize intergenic region and make a nick on this plasmid. And then gp5 dimers will package the single strand of pBluescript because they believe this is their “genome”! In this situation, host cell will release phages but these phages do not contains their genome, they will carry phagemid instead. So they will not be able to make next generation after infection, so we call them “phagemid-carrying phage”. |
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| Fig.3 |
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