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.