Development and optimization of Lysis gene E as a counter-selection marker with high selection stringency

Seamless modification of bacteria chromosome is widely performed both in theoretical and in practical research, for this purpose, excellent counter-selection marker genes with high selection stringency are needed. Lysis gene E from bacteriophage PhiX174 was developed and optimized as a counter-selection marker in this paper. Lysis gene E was firstly constructed under the control of pL promoter. At 42 °C, Lysis gene E could effectively kill Escherichia coli. Seamless modification using E as a counter-selection marker also successfully conducted. It also works in another Gram-negative strain Serratia marcescens under the control of Arac/PBAD regulatory system. Through combining lysis gene E and kil, the selection stringency frequency of pL-kil-sd-E cassette in E. coli arrived at 4.9×10−8 and 3.2×10−8 at two test loci, which is very close to the best counter-selection system, inducible toxins system. Under the control of Arac/PBAD, selection stringency of PBAD-kil-sd-E in S. marcescens arrived at the level of 10−7 at four test loci. By introducing araC gene harboring plasmid pKDsg-ack, 5- to 18- fold improvement of selection stringency was observed at these loci, and a surprising low selection stringency frequency 4.9×10−9 was obtained at marR-1 locus, the lowest selection stringency frequency for counter-selection reported so far. Similarly, at araB locus of E. coli selection stringency frequency of PBAD-kil-sd-E was improved to 3×10−9 after introducing plasmid pKDsg-ack. In conclusion, we have developed and optimized a newly universal counter-selection marker based on lysis gene E. The best selection stringency of this new marker exceeds the inducible toxins system several fold.

Qualitative and quantitative assessment of genotoxins using SRRz lysis reporter under the control of a newly designed SOS responsive promoter in Escherichia coli

A new bacterial genotoxicity detection strain was constructed, in which the cell lysis gene of SRRz from a lambda phage was controlled by a new designed SOS responsive element, designated as Escherichia coli BL21/pUC-PST.

Characterization of a Novel Inactivated Salmonella enterica Serovar Enteritidis Vaccine Candidate Generated Using a Modified cI857/λ PR/GeneEExpression System

ABSTRACTA new strategy to develop an effective vaccine is essential to control food-borneSalmonella entericaserovar Enteritidis infections. Bacterial ghosts (BGs), which are nonliving, Gram-negative bacterial cell envelopes, are generated by expulsion of the cytoplasmic contents from bacterial cells through controlled expression using the modified cI857/λ PR/geneEexpression system. In the present study, the pJHL99 lysis plasmid carrying the mutated lambda pR37-cI857 repressor and PhiX174 lysis geneEwas constructed and transformed inS. Enteritidis to produce a BG. Temperature induction of the lysis gene cassette at 42°C revealed quantitative killing ofS. Enteritidis. TheS. Enteritidis ghost was characterized using scanning and transmission electron microscopy to visualize the transmembrane tunnel structure and loss of cytoplasmic materials, respectively. The efficacy of the BG as a vaccine candidate was evaluated in a chicken model using 60 10-day-old chickens, which were divided into four groups (n= 15), A, B, C, and D. Group A was designated as the nonimmunized control group, whereas the birds in groups B, C, and D were immunized via the intramuscular, subcutaneous, and oral routes, respectively. The chickens from all immunized groups showed significant increases in plasma IgG and intestinal secretory IgA levels. The lymphocyte proliferation response and CD3+CD4+and CD3+CD8+T cell subpopulations were also significantly increased in all immunized groups. The data indicate that both humoral and cell-mediated immune responses are robustly stimulated. Based on an examination of the protection efficacy measured by observations of gross lesions in the organs and bacterial recovery, the candidate vaccine can provide efficient protection against virulent challenge.