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