The complex transposon Tn7 has several features that can be adapted for genetic tool development. In the present study, the high-specificity, high-efficiency pathway targeting attTn7
is the aspect of interest. This pathway, directed by the target selector protein TnsD together with the TnsABC catalytic assembly, mediates insertion into one (E. coli, Yersinia, Acinetobacter, Francisella
) or a small number (Burkholderia
) of sites in a broad array of bacteria (28
). Even human DNA has a high-efficiency site, also near a gene encoding N
-acetyl glucosamine synthase (29
). This highly conserved gene (glmS
in bacteria) provides the widely distributed recognition site, but since the site of actual insertion is outside the coding region, downstream of the recognition site, insertion is well-tolerated.
The arrangement presented here mitigates some of the problems associated with using the original pGRG36 system. Genes within the mobile element are insulated from expression signals originating outside of it, and the high-efficiency cloning approach reduces problems associated with manipulation of large plasmids.
High efficiency insertion allows screening (examination one-by-one) rather than selection (using drug selection to eliminate unproductive background) for the construct desired. The mobile element mTn7(MCS MS26), carried on pMS26, still retains an insertion efficiency high enough to allow screening rather than selection for chromosomal insertions (10% of 42°C survivors; ). Although ampicillin is used to select for the presence of the initial construct, that construct is efficiently lost at 42°C, and drug-sensitivity is restored in the final strain. The streamlined insertion protocol was effective in two host strains of very different pedigrees, with three constructs of distinct function. Possibly the insertion efficiency might be further increased by adding arabinose to the ampicillin plates used to select transformants (Step 4 of ).
The mobile element mTn7(MCS MS26) presumably could also be mobilized via the TnsE-dependent random insertion pathway that targets conjugal plasmids. We have not tested this lower efficiency transposition pathway. TnsE is not encoded on pMS26 but could be provided in trans.
We compared the rhaBp
expression system to the well-characterized lac
expression system in several ways. One trial () examined lacI-
regulated expression of the lacZ
transcription unit, in the context of the mTn7(MCS MS26)
element. In this situation lacZp
directed transcription and was regulated by LacI encoded on the ϕ80 Δ(lacZ)M15
prophage resident in the host. The mTn7Φ(rhaBp-lacZp-lacZ)1
element contains, downstream of rhaBp
, a segment of wild-type lac
operon sequence extending from the end of lacI
to the beginning of lacY
, including all annotated regulatory elements. In the absence of rhamnose, IPTG mediates a 100-fold induction, similar to induction seen in the wild-type lac
operon (not counting glucose repression, not measured here) and in other lacZ
reporter controls [see, e.g. (30
was examined in that case].
Also notable was the lack of rhamnose induction above the background expression from uninduced lacp. The rhaBp transcription start is 31 bp from the closest (furthest upstream of lacp) LacI binding site. Several mechanisms by which bound regulators could interfere with each other or with RNAP can be imagined.
The provision of a tightly controlled inducible rhaBp
promoter together with a strong translation initiation region adds flexibility. Expression is very low in the presence of glucose [, lines 4–9 and lanes (G, +)], indistinguishable from the negative control (, lines 1–3; , lanes (G, –; R, –)]. The highest expression was achieved by combining the strong translation signal (LTS) in M9 medium with rhamnose as sole carbon source (, lines 10–12; , LTS; , M9, R, +). The level of β
-galactosidase activity was ~10% of fully induced lacZ
from its own promoter (compare , IPTG with , lines 10–12). For reasons we have not explored, growth under these conditions is slow, and final cell yields are half that with glucose as carbon source. Induction of the rhamnose operon is known to be slow (31
), possibly accounting for the long growth lag.
Intermediate expression levels can be achieved by combining rhamnose with other nutrients (M9 glycerol
rhamnose, , lines 16–18; rich
rhamnose, , lanes rich, R, +).
The donor plasmid pMS26 also was designed to accommodate alternative cloning and expression strategies, as described in the section on vector design. Such modified protocols will still enjoy the insulation from external expression and the high insertion specificity of the mobile element described here.