The mutagenesis procedure essentially comprises four steps:
The target-specific changes needed to be made to the ClosTron RNA-encoding region are determined using the Perutka method [2].
The small size (344 bp, inclusive of the HindII and BsrGI recognition sites) of the targeting regions identified by Peruka are easy and inexpensive to synthesise. However, the sequence given by the design tool is only 309 bp to suit the strategy used by ATUM to clone the fragment. Alternatively, primers are also given which can be used to PCR amplify the re-targeted region using a method detailed in the first ClosTron paper [3].
ATUM Inc. can rapidly construct your intron targeting region and clone it into your choice of ClosTron plasmid, which they hold in stock.
Your sequence authenticated re-targeted ClosTron plasmid will typically arrive within 2 weeks of ordering, and you will be ready to start the next step.
If you haven't used the ClosTron before, you need to arrange an MTA with The University of Nottingham before you can receive ClosTron plasmids.
Following receipt (ATUM), or construction, of your plasmid(s), introduce into your target organism by either conjugative plasmid transfer or transformation selecting for the plasmid marker.
This will usually be catP, therefore requiring selection on media containing thiamphenicol. Typical transfer procedures are downloadable here:
Constitutive expression of the intron means that intron insertion into the host target begins to take place as soon as the ClosTron plasmid enters the cell. To isolate merely re-streak one or more transformants onto agar plates supplemented with erythromycin (or lincamycin) at a concentration appropriate to the strain. To confirm insertion of the intron in the erythromycin or lincamycin resistant colonies PCR screening will be necessary using primers that flank the intended site of insertion. DNA fragments generated should be authenticated by size and correct nucleotide sequence. To confirm single insertion of the intron it will be necessary to either perform a Southern blot or deploy nanopore whole genome sequencing.