pMTL80000 Series Information

 

pMTL80000 Series

Modular shuttle vectors allow a systematic approach to plasmid design and construction. The pMTL80000 series contains 18 modules flanked by four rare type II restriction sites. These oplasmids are utilised by CHAIN in its own research, and are available to other researchers in the field. A kit of all modules for the pMTL80000 series can be purchased exclusively via CHAIN.

Recombinant plasmids are usually constructed and cloned via E. coli bacteria before being transferred (‘shuttled’) into the production organism of choice. These shuttle plasmids therefore need to include all the features required for replication and maintenance in both hosts as well as an optional transfer function for a conjugation donor strain.

The system is:

  • Modular thus facilitating the combinatorial construction of ClostridiumE. coli shuttle plasmids from standard components of certain types
  • Easy and fast to use being both conceptually and practically simple and requiring a minimum of labour
  • Reversible whereby it is possible to replace standard components of existing modular plasmids after their construction, without the need to re-build the entire plasmid from scratch
  • Extensible in that addition of one or more components outside the standard types is straightforward

Choosing a combination of modules

Replicons, selectable markers, and application-specific modules have been incorporated into the kit. The pMTL80000 series is a standard arrangement in which every plasmid contains exactly one module of each of four types, always arranged in the same order, and always bounded by the same four rare (8 bp) type II restriction enzyme recognition sites. This system allows for the quick and easy modification of existing pMTL80000-based plasmids.

The modular system was developed by the Minton group at the University of Nottingham, and is published (Heap et al, 2009). The system has been extensively tested at the University of Nottingham and continues to be expanded. An updated publication for the Minton lab outlines up-coming technologies available for the pMTL80000 series as well as other modular plasmids (Minton et al, 2016).

Data on the practically-relevant functional properties of the Gram+ replicons and antibiotic markers used in the pMTL80000 series are described in the paper. Using these results and the pMTL80000 system, you can now quickly construct shuttle plasmids for particular hosts and applications using rationally-chosen combinations of modules. Use this handy sequence generator provided by the University of Nottingham to construct a FASTA seqeunce of your vector of interest. The annotated sequences are generated in Genbank (.gb) format. You can open these files using many molecular biology / bioinformatics tools such as GENtle, ApE and others, including some in this list. When viewing the sequences of pMTL80000 plasmids, set your software to show the following restriction sites: AscI, FseI, PmeI, SbfI, ApaI, NotI, NdeI and NheI.

Due to the standard construction, the sequence of pMTL80000 plasmids is precisely defined by the choice of modules, so can easily be determined automatically. Plasmid names are determined by the combination of modules: “pMTL8” followed by the numbers of the combination of modules used, in the correct order.

Components of the pMTL80000 Series Kit

Gram+ replicon Marker Gram replicon Application-specific
0. spacer 1. catP 1. p15a 0. spacer
2. pBP1 2. ermB 2. p15a + tra 1. MCS
3. pCB102 3. aad9 4. ColE1 2. Pthl + MCS
4. pCD6 4. tetA(P) 5. ColE1 + tra 3. Pfdx + MCS
5. pIM13 4. catP reporter

References

  1. Heap, J.T. et alA modular system for Clostridium shuttle plasmidsJ Microbiol Methods. 78, 79-85 (2009).
  2. Minton, N.P. et alA roadmap for gene system development in Clostridium. Anaerobe. 41, 104-112 (2016).