Directed mutagenesis is an important approach in molecular microbiology, but has historically proven difficult in many Clostridium species. Recently we developed the ClosTron system, which allows the directed construction of stable mutants in Clostridium species using a bacterial group II intron. These are broad host-range elements whose target specificity is determined largely by base-pairing between intron RNA and target site DNA. Such introns can therefore be rationally re-targeted by altering the sequence of an intron RNA-encoding plasmid. The ClosTron system (like most group II intron approaches) uses an element derived from the broad host range Ll.LtrB intron of Lactococcus lactis.
The ClosTron method is optimised to take full advantage of some important characteristics of group II introns, making mutagenesis in Clostridium easy:
ClosTron plasmids provided already targeted to your gene and ready to use
Intron target specificity is determined by a small region, so it is cost-effective to re-target an intron by out-sourcing the synthesis and sub-cloning of the small DNA fragment. ClosTron plasmids are delivered ready-targeted, typically within 2 weeks of ordering.
Rapid integrant isolation
Once the ClosTron plasmid is transferred into the host organism, the intron is constitutively expressed and spontaneously inserts into the chromosome in some cells in the population. Thanks to the RNA-mediated mobility of the intron and a special type of marker called a RAM, the clones with an intron insertion become resistant to a second antibiotic, allowing them to be easily isolated. This powerful selection is generally-applicable – it does not depend upon properties of the target gene.
The intron uses a replicative mechanism (i.e. copy-and-paste not cut-and-paste) and is delivered using a mini-intron arrangement on a ClosTron plasmid, which is later lost. Together, these properties ensure that constructed strains are stable.
The mutagenesis procedure is logically separated into four steps: intron design, plasmid construction, plasmid transfer, and integrant isolation. These are described below.
Intron target site recognition by Ll.LtrB-derived introns is more complex than the simple sequence identity which homologous recombination depends upon. Determining the sequence of an intron modified to insert into your gene of interest is therefore slightly more involved than designing a conventional allele exchange cassette. Fortunately, this step lends itself very well to automation. The intron design tool provided here guides you through the design in a few minutes, performing all the bioinformatics automatically.
In Step 1 (intron design), you designed an intron targeting region for a site within your gene of interest. This targeting region is short - only 344 bp, inclusive of the HindII and BsrGI site (BUT Note, the DNA sequence given by the design tool is only 309 bp to suit the strategy used by DNA2.0 to clone the fragment) so it is cheap to construct synthetically. DNA 2.0 Inc. can quickly construct your intron targeting region and clone it into your choice of ClosTron plasmid, which they hold in stock. They also check the sequence is error-free. Your re-targeted ClosTron plasmid will typically arrive within 2 weeks of ordering, and you will be ready to start Step 3 (plasmid transfer) immediately.
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. See the legal stuff section below.
Please read the brief instructions before ordering, especially if you have not ordered a re-targeted ClosTron plasmid from DNA 2.0 before.
Current pricing: US$175 per re-targeted plasmid, US$50 shipping charge per order.
Note – We have no financial interest in DNA 2.0, and we don't get any money when you order a re-targeted ClosTron plasmid from them. We started this service because we think it is the best way to re-target introns, and it is cost-effective.
Previously described methods re-target the intron by cloning a DNA fragment generated by SOE PCR. This is a similar approach to the construction of a typical allele exchange cassette, and requires a similar amount of effort. If you prefer, you may still use the original SOE PCR intron re-targeting procedure. A PCR protocol is provided on page 6 of the TargeTron user guide. The one-tube SOE PCR protocol requires a special PCR template. You can obtain a suitable template as part of the TargeTron kit, or by contacting us.
When you have obtained a suitable ClosTron plasmid re-targeted to your chosen target site, transfer the plasmid into the host organism using a standard method. Select transformants or transconjugants containing the ClosTron plasmid using an antibiotic selection suitable for the plasmid and host organism. For example, cells containing ClosTron plasmid pMTL007C-E1 or pMTL007C-E2 are selected using chloramphenicol or thiamphenicol, while cells containing ClosTron plasmid pMTL007S-E1 or pMTL007S-E2 are selected using spectinomycin. Suitable antibiotic selection conditions vary between species and strains, but some useful antibiotic working concentrations are given in the pMTL80000 paper.
Organisms into which plasmids are typically transferred by conjugation:
Clostridium difficile, Clostridium botulinum, Clostridium sporogenes, Clostridium beijerinckii, Clostridium novyi, Clostridium sordellii, and others.
Organisms into which plasmids are typically transferred by electroporation:
Clostridium acetobutylicum and Clostridium perfringens.
Intron expression from ClosTron plasmids is constitutive, and starts as soon as the ClosTron plasmid has been transferred into the host cell. This means that when transformant or transconjugant colonies become visible after transfer of the ClosTron plasmid, there will already be some integrant cells (cells in which the intron has integrated into the chromosome) present within each colony. To isolate integrant clones from the mixture of cells in these colonies, simply re-streak one or more transformant / transconjugant colonies onto agar plates containing erythromycin or lincomycin at a concentration to which the parental strain is sensitive. Usually these can be re-streaked directly from the transformation or conjugation selection plates, without first isolating transformant / transconjugant clones in the typical way. If re-streaking directly from conjugation selection plates, be sure to include the conjugation donor counter-selection agent in the erythromycin / lincomycin plates.
To screen erythromycin-resistant / lincomycin-resistant clones for the desired insertion you will need specific primers which flank your target site. You should design, obtain and validate these primers using the wild-type / parental strain. If you don't already have a preferred method for primer design, consider trying Primer3, Primer3Plus, or other options from this list.
We provide ClosTron plasmids free-of-charge. However, we are obliged to provide ClosTron plasmids only under certain terms and conditions. These define some rights and responsibilities, essentially to protect the intellectual property associated with the ClosTron, particularly when it is used in commercial research (the MTA has almost no effect on purely academic research). To formalise these rights and responsibilities, there must be a Material Transfer Agreement (MTA) in place between your institution and The University of Nottingham. Until an MTA is in place, neither we nor DNA 2.0 can provide you with ClosTron plasmids.