Potential of lysis gene E as a counter-selection marker gene
Several previous studies have reported that expression of lysis geneE could promote the death of many Gram-negative bacteria through
fusion of the inner and outer membranes. These results show that the
product of lysis gene E has a universal killing effect on
Gram-negative bacteria, which prompt us to explore the potential of
lysis gene E being a generic counter-selection marker gene for
genetic modifications in Gram-negative bacteria. For this purpose,
expression of lysis gene E should be controllable. In our
previous work, kil counter-selection cassette under the control
of promoter pL function well in E. coli (Wei Chen et al.,
2019). We want to test the killing effect of lysis gene E under
the same condition. Therefore, plasmid pBBR1-MC5 and the CDS (Coding
Sequence) of lysis gene E were firstly ligated after digestion
with Sph Ⅰ and Nco Ⅰ.
Thus, the CDS of lysis gene E is linked with the gentamycin
resistance gene GmR in the newly constructed
plasmid pBBR1-E (Fig. 1A). Using dsDNA mediated Red homologous
recombination, PCR amplified E-GmR (including
gentamycin resistance gene and CDS of lysis gene E) cassette was then
used to substitute the kil CDS of tet-kil double selection
cassette of E. coli MG-10 (Figure 1B). The goal strain, named as
CWE-1, was successfully constructed (Fig 1C). To exclude the
interference of GmR , tet-pl-E cassette
was amplified and used to substituted the CDS of ack . The goal
strain was names as CWE-2 (Fig. 1D).
According to our expectation, since expression of lysis gene E in
CWE-2 is driven by the pL promoter, which is in turn under the
control of temperature-sensitive cI857 repressor (Wei Chen et al., 2019;
Yu et al., 2000), lysis gene E could be induced
because of the loss-of-function of
the repressor cI857 at 42 °C. If expression of lysis gene E 42 °C
could effectively cause the death of host cell, it has the potential of
being a counter-selection marker. As shown in Fig. 2A, after streak
culture no growth traces of CWE-2 can be found at LB agar plate placed
at 42 °C. This is similar to the positive control strain MG-10, in which
counter-selection marker gene kil constructed was induced to kill
host strain (Wei Chen et al., 2019). At the same time, obvious growth of
the negative control strain MG1655 [pSim6] appeared on agar plate.
In contrast, all strains grew well at 30 °C. It indicates that our newly
constructed lysis gene E expression cassette meet the requirement
of a negative-selection marker gene: having no negative influence in the
growth of host cell under non-induced conditions, while effectively
killing its host under induced conditions. This is also verified by spot
titers experiment (Fig. 2B). Our results indicate that lysis geneE has good potential to be a counter-selection marker.
Seamless modification testing of thelysis gene E
counter-selection cassette in E.coli
To directly investigate the effect of lysis gene E in
counter-selection, PCR amplified
1,000 bp DNA fragment ofcatenin gene from Helicoverpa armigera with 38 bp of short
homologous arms was used to substitute the foreign tet-pL-E at
the ack locus of CWE-2. At 42 °C sparsely spread colonies were shown in
the control group which was electroporated without adding DNA, whereas
much more colonies appeared in the group electroporated with dsDNA (Fig.
2C). The following colony PCR showed that 9 out of 10 randomly selected
colonies were all correct recombinants (Fig. 2D). These results suggest
that seamless modification using lysis gene E as a
counter-selection marker is feasible in E. coli .
Application of