Discussion
Classic genomic modification to knockout out genes is usually conducted
by inserting antibiotic resistance markers. This is simple and easy to
implement. However, the existence of unwanted antibiotic resistance
marker genes may leave some unexpected effects, which is not expected by
researchers in metabolic engineering and functional genome analysis.
Through two-step selection/counter-selection method, target genes can be
edited without alerting other parts of the chromosome. This unmarked
mutation, known as “seamless modification”, overcomes the
above-mentioned shortcomings.
Although many counter-selection markers have been developed, such assacB , ccdb , I-SceI , galk, mazF (Z. Chen,
Ling, & Shang, 2016; DeVito, 2008; Muyrers et al., 2000; Van Zyl,
Dicks, & Deane, 2019; Wang et al., 2014; Warming, Costantino, Court,
Jenkins, & Copeland, 2005) etc., excellent markers are still limited.
Counter-selection genes suffer from some problems which limit their
application in genomic modification, among which high background is a
serious obstacle. For example, although sacB is most widely used,
researchers have to face low positive rates due to the spontaneous
mutations that inactivate sacB (Khetrapal et al., 2015; Y. Zhang,
F. Buchholz, J. P. Muyrers, & A. F. J. N. g. Stewart, 1998). Therefore,
low background is an important consideration when counter-selection
genes are to be choose.
Selection stringency frequency is an indicator that shows the level of
background during counter-selection. It has a crucial role of
determining the ratio of correct recombinants in counter-selection (Wei
Chen et al., 2019). Selection stringency is reflecting by calculating
the fraction of viable colonies that escape the selection stress. The
smaller the number is, the higher the stringency is (Khetrapal et al.,
2015). So far, inducible toxins system performs best among all the
developed counter-selection system, followed by tetA-sacBcassette. As for toxins system, the best selection stringency frequency
achieved in E. coli is 1.11×10−8, which is
exceeds the next best reported tetA–sacB system up to 60- fold.
The selection stringency frequency even approaches that of kanamycin
resistance gene (2.4×10−9) (Khetrapal et al., 2015).
Excellent counter-selection markers like inducible toxins system will
greatly improve the positive rates and avoid wasting time on redundant
work during genomic seamless modification.
Inspired by the inducible toxins system, in this paper we developed a
powerful generic counter-selection system for genomic modification,
especially for some species which lack efficient genetic tools. Lysis
gene E is of general killing effect on Gram-negative bacteria.
Given the speculation that counter-selection based on a universal lethal
gene would be applicable generally. We focused our attention on the
developing and optimizing of lysis E as a counter-selection
marker. Expression of lysis gene E under the control of pLpromoter efficiently killed its host. dsDNA mediated seamless
modification were also successfully performed to substitute target
region, and the ratio of correct recombinants is higher than kilcounter-selection marker constructed previously in our lab (Wei Chen et
al., 2019). In S. marcescens , lysis gene E also can be
used for counter-selection marker both through ssDNA and dsDNA mediated
recombination. However, the ratio of correct recombinants through dsDNA
mediated recombination is lower than that in E. coli . Maybe the
difference in ratio of correct recombinants can be attributed to
different gene expression regulation system used for controlling lysis
gene E . After all, more lysis protein E could be produced
to kill bacteria under the control of strong promoter pL compared
with rigorous PBAD promoter.
Lysis gene E could be used as a counter-selection marker both inE. coli and in S. marcescens. Since it has general lethal
efficiency in Gram-negative strains, it is also applicable in other
Gram-negative strains as long as promoter pL andPBAD could work. Although a universal
counter-selection gene based on E was developed by us, there
still existed escaping colonies during seamless modification, especially
in S. marcescens. This leads to relative low ratio of correct
recombinants during PCR identification when dsDNA mediated recombination
was conducted in S. marcescens . Of course escaping colonies also
existed in ssDNA mediated recombination, high ratio of correct
recombinants is attributed to the high recombination efficiency of ssDNA
itself (Ellis, Yu, & DiTizio, 2001). It should be pointed out that in
order to make the colonies exhibit clearly in the LB plate, only 5μl of
recovered cells after transportation was plated in ssDNA recombination,
while 100 μl was plated in dsDNA recombination.
To provide a good alternative tool for seamless modification,
counter-selection system based on lysis gene E need to be
optimized to improve its selection stringency. In our previous work, we
developed a counter-selection cassette based on kil gene of
lambda phage. It performs well in E. coli and its selection
stringency is comparable to the next best counter-selection systemtetA -sacB . Both kil gene and E gene are
short in length: the CDS of kil and E is 144 bp and 273 bp
separately. We believe that combining kil and E gene in a
certain way would be a good idea. An obvious benefit is that the
selection stringency would be greatly improved. What is more, it will
not bring any additional difficulties to the experimental process. After
all, CDS length of the counter-selection cassette after combining the
two genes is less than 500 bp, which is shorter than most of the
counter-selection markers. We have tried to combine the two gene through
fusion expression, however no selection stringency improvement was
observed (data now shown). Maybe the function of proteins was affected
by the changes in spatial conformation after fusion expression. We think
co-express the two genes in the form of bi-cistron should be a better
alternative solution from the perspective of reducing the background.
Because nonsenses mutation in the first gene would not inactivate the
second gene since independent products are translated, which is not
applicable for fusion expression. Co-express kil and Egene through a classic RBS between their CDS indeed greatly improved the
selection stringency to the level of the best reported toxins inducible
system in E. coli (4.9×10−8 at ack locus
and 3.2×10−8 at araB locus). Surely, it is
possible to obtain better selection stringency through combiningkil and E in other ways, such as improving translation
initiation of the second gene using “bicistronic design” (BCD)
reported by Mutalik (Mutalik et al., 2013). Many other strategies could
also be used, such as combining more counter-selection markers with
short size in length. Our work, in this paper, mainly aims to provide an
example for improving selection stringency based on existing markers.
Other than developing the high selection-stringent kil-sd-Ecounter-selection cassette under the control of pL promoter in
this paper, we also constructed AraC/PBAD controlling kil-sd-Emarker which could be used in bacteria where high temperature is not
suitable for growth. In S. marcescens , it does not perform as
excellent as pL promoted kil-sd-E . By introducingaraC gene harboring plasmid, the selection stringency was
improved 4- to 170- fold. In particular, the selection stringency at
these loci tested in this paper is all comparable to the best
counter-selection system, inducible toxins system. At araB locus
in E. coli and marR-1 locus in S. marcescens , it
reaches the level of
10−9, the lowest selection stringency frequency for
counter-selection so far.
This gives us an enlightenment: we could integrate the regulating
elements of counter-selection markers, such as araC gene, into
Red recombinases genes harboring plasmid. Therefore, counter-selection
cassette will be reduced greatly in size. Take
AraC/PBAD-kil-sd-E-GmR double
selection cassette as an example, if araC gene was integrated
into the Red recombination system providing plasmid, only 1,327 bp
PBAD-kil-sd-E-GmR cassette was
needed for seamless modification. This will not only facilitate PCR
amplification, but also improve recombination efficiency because longer
substrate dsDNA decreases the chances of a λ Red recombination event
(Wei Chen et al., 2019).