Replicative plasmids are basic tools in molecular microbiology. Many experimental approaches start with the transfer of a recombinant plasmid into the strain of interest. Cells containing the plasmid are then typically selected and maintained using an antibiotic resistance marker encoded by the plasmid, by adding a suitable antibiotic to the growth medium. For practical reasons, recombinant plasmids are usually constructed and stored in cloning strains of E. coli, then transferred (‘shuttled’) into the organism of interest in order to perform experiments. These shuttle plasmids therefore need to include all the features required for replication and maintenance in both hosts. Shuttle plasmids are often transferred by conjugation, and in these cases the plasmid must also include a transfer function compatible with the donor strain.
Several shuttle plasmids have been used in Clostridium previously, each having different functional properties and host ranges. The plasmids do not share a common structure, reflecting their construction in different labs, probably without much emphasis on a design that can easily be modified. Consequently, altering these plasmids tends to require ad-hoc cloning strategies which may prove difficult and time-consuming. Furthermore, it is not possible to directly compare the differing functional properties of these plasmids.
This led to some problems, especially for a group like ours which works on several different Clostridium species:
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.
Once we had established the pMTL80000 system, we were finally able to generate directly-comparable data on the practically-relevant functional properties of the Gram+ replicons and antibiotic markers we use. These studies are described in the paper. Using these results and the pMTL80000 system, we (and you) can now quickly construct shuttle plasmids for particular hosts and applications using rationally-chosen combinations of modules.
In order to distribute the 18 modules in a convenient way, we constructed the first five plasmids shown in the table below which, between them, contain all 18 modules. This kit, with the additional four plasmids containing each Gram+ replicon as shown in the table, is now available from CHAIN Biotech. CHAIN will oversee the distribution of the pMTL80000 series from 1st December 2015. From the kit, the other 395 possible combinations of modules can be easily constructed in one or a few cloning step(s) using AscI, FseI, PmeI and SbfI.Request a kit from CHAIN biotech here.
|Plasmid||Gram+ replicon||Marker||Gram– replicon||Application-specific|
|pMTL80110||0. spacer||1. catP||1. p15a||0. spacer|
|pMTL82254||2. pBP1||2. ermB||5. ColE1 + tra||4. catP reporter|
|pMTL83353||3. pCB102||3. aad9||5. ColE1 + tra||3. Pfdx + MCS|
|pMTL84422||4. pCD6||4. tetA(P)||2. p15a + tra||2. Pthl + MCS|
|pMTL85141||5. pIM13||1. catP||4. ColE1||1. MCS|
|pMTL82151||2. pBP1||1. catP||5. ColE1 + tra||1. MCS|
|pMTL83151||3. pCB102||1. catP||5. ColE1 + tra||1. MCS|
|pMTL84151||4. pCD6||1. catP||5. ColE1 + tra||1. MCS|
|pMTL85151||5. pIM13||1. catP||5. ColE1 + tra||1. MCS|
When we designed the pMTL80000 system and constructed the 18 modules shown here, we incorporated those elements expected to be most useful in our work. We are well aware that other useful elements exist. The current choice of modules is not intended to be definitive or static, indeed the system was designed from the start to be extensible. If you would like to add another useful module to the system in the correct format, and would be willing to share it, please contact CHAIN Biotech.