Improving selection stringency frequency of kil-sd-E by
introducing plasmid pKDsg-ack
Selection stringency frequency ofkil-sd-E under the control of AraC/PBAD system
was at the level of 10−7 at the pigA locus
(Fig. 5H). We analyzed other three loci of S. marcescens (The
first gene code for MarR family transcriptional regulator, we named it
as marR-1 ; the second gene rcsA codes for DNA-binding
transcriptional activator; the last gene code for a hypothetical
protein, we named it as hyp-1 ), the results are similar (Fig.
6A), other than a very low stringency frequency occurred atmarR-1 locus (2.5×10−8). Although selection
stringency frequency of 10−7 is relatively high and
even better than the next best tetA-sacB system in E.
coli , it is still not as good as kil-sd-E counter-selection
marker controlled by pL promoter.
To provide a more powerful generic counter-selection marker, wo want to
elevate the selection stringency of this system. PromoterPBAD is dual regulated by transcription factors
Arac. Arac can bind to PBAD and act as repressor
or inducer depending on the absence or presence of arabinose. The
increase in the number of transcription factor AraC can not only promote
the expression of gene it controlled after addition of arabinose, but
also reduce leakage expression of gene it controlled in de absence of
inducer. Therefore, we suspected that the existence of multiplearaC genes is very likely to be beneficial to the enhancement of
selection stringency.
Plasmid pKDsg-ack which carried the araC gene (Reisch & Prather,
2015) was introduced into the host cell after the original plasmid pSim6
being cured under 37°C cultivation. In MG1655, selection stringency
frequency of kil-sd-E at araB locus in pKDsg-ack harboring
strain decreased surprisingly to 3×10−9, This is the
lowest selection stringency frequency reported so far. And it is about
170- fold lower than that of pSim6 harboring strain (Fig. 6B). It must
be pointed out that plasmid pKDsg-ack is not
lethal to host in the presence of
arabinose (Fig. S4).
In S. marcescens , selection stringency frequency ofkil-sd-E reached 10−8 to 10-9at all these loci, about 10- fold lower than before. In detail, the
selection stringency frequency at the pigA , rcsA , andhyp loci was 3.1×10−8,
4.7×10−8 and 2.5×10−8 (Fig. 6C). A
surprisingly low selection stringency frequency,
4.8×10−9 (Fig. 6C), occurred at the mar locus.