Phagemid pBluescript is a special plasmid, it functions as a normal plasmid when transformed into bacteria. But when helper phage M13KO7 infect the bacteria, it will produce progeny M13 phage as vector for pBluescript.
pBluescript contains a f1 ori while M13KO7 helper phage’s f1 ori has been knock-down. So after the infection of M13KO7, its genome can produce protein and replicate but these proteins will package pBluescript as their true “genome”. As a result, the progeny phage’s genome will only contains pBluescript’s MCS, they cannot make next generation.
Source : XVIVO/Barcroft USA
BBa_K1473009
Here we combined a CRISPR system(BBa_K1218011) with an inducible promoter(BBa_K914003). The CRISPR system contains a tracrRNA , a Cas9 protein and a minimal
CRISPR array, it functions to knockout target gene. We give this system an inducible promoter so we can decided when to switch if on or off.
We put a sacI restriction cutting site downstream to gRNA (not shown in sequence information) and ligated it back to pSB1C3 with correct prefix and surfix. Then we cut this recombinant plasmid with sacI and run gel electrophoresis for testing. As we can see in this image, because pSB1C3 only contains 1 sacI cutting site itself, so it would not be cut into 2 parts when no gRNA is ligated inside. But if there is, then sacI will cut this recombinant plasmid into 2 parts, whose lengths are 939 bp and 1159 bp. Obviously, we have successfully synthesized this gRNA.
Parts Table
Any parts our team has created will appear in this table below:
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BBa_K1473005
BBa_K1473009
