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pMTL80000 modular plasmids

Introduction to shuttle plasmids
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.

Shuttle plasmids in Clostridium

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:

We wanted to construct shuttle plasmids most suitable for our research, but did not have data on
which combination of elements would be best, nor did we have directly-comparable plasmids
which would allow us to generate such data.
Even if we knew which combination of elements would be most suitable, the shuttle plasmids
could not be constructed quickly, reliably or in a consistent way.
If our first choice of elements proved not to be suitable, or when we wished to adapt a useful
plasmid for a different host or application, it was not necessarily easy to modify the plasmid, and
the new combination of elements might mean an entirely new cloning strategy.

A more systematic approach pMTL80000 modular shuttle plasmids

Eventually we decided to invest some effort in establishing a systematic approach to shuttle plasmid
construction, hoping it would save us time in the long run (fortunately, it has). The pMTL80000 modular
plasmid system was the result of this effort, and is published.

Get the pMTL80000 paper

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Essentially, we defined 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. The standard
arrangement is shown in the plasmid
diagram. We also constructed a total of 18
modules of these four types, flanked by
the appropriate restriction sites. The
modules are numbered, and are listed
below. This system allows the
combinatorial construction of shuttle
plasmids from modules in the standard
format. It also provides for the quick and
easy modification of existing pMTL80000-
based plasmids.

When constructing plasmids using the

existing modules no real design process is
required the researcher need only chose
the desired combination of modules from the available selection. 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 also determined by the combination of modules: pMTL8
followed by the numbers of the combination of modules used, in the correct order. Consequently, the
short plasmid names each conveniently imply a precise sequence. To download the annotated sequence
of a pMTL80000 plasmid, just select one module of each type from the menu below, then click the
Generate and download button.

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

Generate and download

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.

Choosing a combination of modules

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
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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.

Kit for distribution of the pMTL80000 system

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

Expanding the system

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.

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