Allele Coupled Exchange (ACE) allows the creation of complementation and overexpression mutants in Clostridium spp. ACE vectors may be used to introduce a DNA fragment of up to 28 kb, a prerequisite for the engineering of strains to contain new functions and pathways. All clostridia transformed to date contain the required pyrimidine pathway, making this technique universally applicable to drive the industrial exploitation of clostridia.
ACE is compatible with strains harbouring truncated pyrE, a gene in the pyrimidine synthesis pathway, thus creating a mutant which is both uracil auxotrophic and 5-fluoroorotic acid resistant. These Clostridium strains, available from CHAIN, are:
- Clostridium acetobutylicum ATCC 824 ΔpyrE
- Clostridium difficile 630 ΔpyrE
- Clostridium difficile R20291 ΔpyrE
This pyrE mutant strain forms the basis for all future mutations using the ACE system.
A suite of ACE vectors are now available for this purpose.
These vectors are:
ACE Repair: Restore the pyrE gene at natural locus
ACE Complementation: Complement a knock-out mutation
ACE Expression: Overexpress a gene of interest with a strong promoter
In all cases, the pyrE gene is concomitantly restored at its locus and integration is simply selected by restoration of uracil prototrophy. The complementation function works with knockout strains generated with CHAIN’s pMTL80000 Series Vectors.
The availability of the pMTL80000 series has allowed the formulation of a simple ‘roadmap’ for gene system development in clostridial species.
The system revolves around the creation of a truncated pyrE mutant. Such mutants are both uracil auxotroph and resistant to 5-Fluoroorotic acid. The pyrE mutant forms the basis of all future manipulations using any mutagen. This is, because regardless of the mutagen employed, the pyrE mutation can be complemented concurrently.
Further to the pyrE mutant strains, CHAIN also supplies cloning, conjugation donor and methylation strains needed for successful implementation of the roadmap.
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