Fig 1 - uploaded by Monthon Lertcanawanichakul
Content may be subject to copyright.
Restriction map of the shuttle plasmid vectors generated and utilized in this study (Table 1). The vectors were based on the original lactococcal plasmid pIL253 (A). Then, it was used to construct the Escherichia coli-enterococci shuttle vector pOri253 (B) by inserting the oriColE1 replicon at the XbaI restriction site of its multicloning sites (MCS). The shuttle vector pOri253 was further modified by inserting lactococcal promoter P23 at the EcoRI/BamHI end of the MCS, resulting in the lactococcal expression vector pOri23 (C). Finally, the P23containing plasmid vector was used to subclone the bacteriocin N15 (entAI) gene to be expressed in enterococci (D). Em r is the erythromycin-resistant gene, and repD and repE are the genes responsible for replication in either enterococci or lactobacilli. The arrow indicates the direction of transcription.
Source publication
A 6.09-kb plasmids vector pOri253 was constructed from the plasmid pIL253 (5.2 kb) and a 0.89-kb fragment of oriColE1 from pBluescript II KS. The bifunctional plasmid pOri253 conferred erythromycin resistance in both Escherichia coli and Enterococcus faecalis. It has unique sites for EcoRI, BamHI, SalI, and PstI derived from pIL253 and was lost at...
Contexts in source publication
Context 1
... was amplified by PCR [21], and inserted at the XbaI restriction site of pIL253. The resulting shuttle vector pOri253 (Table 1, Fig 1B) could successfully replicate in both E. coli and Enterococcus faecalis. The plasmid contains four cloning sites of EcoRI, BamHI, SalI, and PstI. To obtain the expression vector, pOri253 was modified by inserting the lactococcal promoter P23 [30]. The promoter was amplified by PCR from the L. lactis chromosome [21] and then inserted at the EcoRI/BamHI site of pOri253 at one extreme of the cloning site, des- ignated as pOri23 (Table 1, Fig. 1C). The constructed plasmid has the ability to replicate both in E. coli and enterococci, and also has shown erythromycin resis- ...
Context 2
... was amplified by PCR [21], and inserted at the XbaI restriction site of pIL253. The resulting shuttle vector pOri253 (Table 1, Fig 1B) could successfully replicate in both E. coli and Enterococcus faecalis. The plasmid contains four cloning sites of EcoRI, BamHI, SalI, and PstI. To obtain the expression vector, pOri253 was modified by inserting the lactococcal promoter P23 [30]. The promoter was amplified by PCR from the L. lactis chromosome [21] and then inserted at the EcoRI/BamHI site of pOri253 at one extreme of the cloning site, des- ignated as pOri23 (Table 1, Fig. 1C). The constructed plasmid has the ability to replicate both in E. coli and enterococci, and also has shown erythromycin resis- ...
Context 3
... of the E. coli-Enterococci Shuttle Vector. The shuttle vector pOri253 (6.09 kb) in Table 1 and Fig. 1 was designed to replicate in both E. coli and enterococci. It was constructed from pIL253, previously described [26], carrying an erythromycin resistant gene (Em r ) which can confer erythromycin resistance to both enterococci and E. coli, including lactococci [16]. However, pIL253 (Fig 1A) does not replicate autonomously in E. coli. To overcome this problem, pIL253 was equipped with the high copy- number oriColEI replicon of pBluescript II ...
Context 4
... of the E. coli-Enterococci Shuttle Vector. The shuttle vector pOri253 (6.09 kb) in Table 1 and Fig. 1 was designed to replicate in both E. coli and enterococci. It was constructed from pIL253, previously described [26], carrying an erythromycin resistant gene (Em r ) which can confer erythromycin resistance to both enterococci and E. coli, including lactococci [16]. However, pIL253 (Fig 1A) does not replicate autonomously in E. coli. To overcome this problem, pIL253 was equipped with the high copy- number oriColEI replicon of pBluescript II ...
Similar publications
A fragment of deoxyribonucleic acid 5,300 base paris long and containing the promoter-proximal portion of the histidine operon of Escherichia coli K-12, has been cloned in plasmid pBR313 (plasmids pCB2 and pCB3). Restriction mapping, partial nucleotide sequencing, and studies on functional expression in vivo and on protein synthesis in minicells ha...
The identification and chromatographic characterization of the leukotoxin of Pasteurella haemolytica is described. The toxin, which has an apparent native molecular weight of greater than 400,000 as judged by gel exclusion chromatography, has a 105-kilodalton (105K) polypeptide as its major protein component. The proteolytic degradation of the 105K...
Kataria, R.S. and Rai, A. 1997. Restriction mapping of HindIII fragment ‘J’ of bovine herpesvirus-1 DNA cloned in opposite orientation. J. Appl. Anim. Res., 11: 183–188.
Cloning of HindIII fragments of bovine herpesvirus-1 DNA in pUC9 plasmid vector yielded recombinants carrying ‘J’ fragment cloned in two opposite orientations in different clones....
Two Pseudomonas strains were engineered to contain the nptII gene and plasmid vector sequences in their chromosomes. After incubation of these strains in nonsterile soil, total bacterial DNA was isolated and analyzed by Southern blot hybridization with the nptII gene and the plasmid vector as probes. In addition to the expected bands of hybridizati...
The mercury resistance (mer) operon of plasmid R100 was cloned onto various plasmid vectors to study the effect of mer gene amplification on the rate of Hg2+ reduction by Escherichia coli cells. The plasmids were maintained at copy numbers ranging from 3 to 140 copies per cell. The overall Hg2+ reduction rate of intact cells increased only 2.4-fold...
Citations
... The GAS strains used in these experiments were all generated in the M1 serotype strain 5448 and are summarized in Table 1 [25][26][27][28][29]. All bacterial strains were stored at −80 • C in THY containing 20% (v/v) glycerol. ...
Streptococcus pyogenes, also known as the Group A Streptococcus (GAS), is a Gram-positive bacterial pathogen of major clinical significance. Despite remaining relatively susceptible to conventional antimicrobial therapeutics, GAS still causes millions of infections and hundreds of thousands of deaths each year worldwide. Thus, a need for prophylactic and therapeutic interventions for GAS is in great demand. In this study, we investigated the importance of the gene encoding the delta (δ) subunit of the GAS RNA polymerase, rpoE, for its impact on virulence during skin and soft-tissue infection. A defined 5448 mutant with an insertionally-inactivated rpoE gene was defective for survival in whole human blood and was attenuated for both disseminated lethality and lesion size upon mono-culture infection in mouse soft tissue. Furthermore, the mutant had reduced competitive fitness when co-infected with wild type (WT) 5448 in the mouse model. We were unable to attribute this attenuation to any observable growth defect, although colony size and the ability to grow at higher temperatures were both affected when grown with nutrient-rich THY media. RNA-seq of GAS grown in THY to late log phase found that mutation of rpoE significantly impacted (>2-fold) the expression of 429 total genes (205 upregulated, 224 downregulated), including multiple virulence and “housekeeping” genes. The arc operon encoding the arginine deiminase (ADI) pathway was the most upregulated in the rpoE mutant and this could be confirmed phenotypically. Taken together, these findings demonstrate that the delta (δ) subunit of RNA polymerase is vital in GAS gene expression and virulence.
... The Gram-positive bacteria can then be transformed with the final plasmid, avoiding more time-and resource-consuming techniques that are necessary for applying molecular biology techniques with Gram-positive hosts. The pOri253 plasmid ( Figure 6B) was constructed by insertion of the ColE1 origin of replication from Gram-negative bacteria in the pIL253 MCS [186]. After the insertion of the P23 lactococcal promoter, the new expression vector was called pOri23 [19]. ...
The Lactococcus lactis bacterium found in different natural environments is traditionally associated with the fermented food industry. But recently, its applications have been spreading to the pharmaceutical industry, which has exploited its probiotic characteristics and is moving towards its use as cell factories for the production of added-value recombinant proteins and plasmid DNA (pDNA) for DNA vaccination, as a safer and industrially profitable alternative to the traditional Escherichia coli host. Additionally, due to its food-grade and generally recognized safe status, there have been an increasing number of studies about its use in live mucosal vaccination. In this review, we critically systematize the plasmid replicons available for the production of pharmaceutical-grade pDNA and recombinant proteins by L. lactis. A plasmid vector is an easily customized component when the goal is to engineer bacteria in order to produce a heterologous compound in industrially significant amounts, as an alternative to genomic DNA modifications. The additional burden to the cell depends on plasmid copy number and on the expression level, targeting location and type of protein expressed. For live mucosal vaccination applications, besides the presence of the necessary regulatory sequences, it is imperative that cells produce the antigen of interest in sufficient yields. The cell wall anchored antigens had shown more promising results in live mucosal vaccination studies, when compared with intracellular or secreted antigens. On the other side, engineering L. lactis to express membrane proteins, especially if they have a eukaryotic background, increases the overall cellular burden. The different alternative replicons for live mucosal vaccination, using L. lactis as the DNA vaccine carrier or the antigen producer, are critically reviewed, as a starting platform to choose or engineer the best vector for each application.
... Kb) derived from pIL253 plasmid. 15 ...
Lactococcus lactis is well documented as a promising candidate for development of novel oral live vaccines. It has been broadly engineered for heterologous expression, as well as for plasmid expression vector delivery, directly inside eukaryotic cells, for DNA vaccine or as therapeutic vehicle. This work describes the characteristics of a new plasmid, pExu (Extra Chromosomal Unit), for DNA delivery using L. lactis and evaluates its functionality both by in vitro and in vivo assays. This plasmid exhibits the following features: i) a theta origin of replication, ii) an expression cassette containing a multiple cloning site and an eukaryotic promoter, the Cyto-Megalo Virus (pCMV). The functionality of pExu:egfp was evaluated by fluorescence microscopy. The L. lactis MG1363 (pExu:egfp) strains was administered by gavage to Balb/C mice and the eGFP expression was monitored by fluorescence microscopy. The pExu vector has demonstrated an excellent stability either in L. lactis or in Escherichia coli. The eGFP expression at different times in in vitro assay shown that 15.8% of CHO cells were able to express the protein after transfection. The enterocytes of mice shown the expression of eGFP protein. Thus, L. lactis carrying the pExu is a good candidate to deliver genes into eukaryotic cells.
Streptococcussuis is an important zoonotic pathogen that causes severe invasive disease in pigs and humans. Current methods for genome engineering of S. suis rely on the insertion of antibiotic resistance markers, which is time-consuming and labor-intensive and does not allow the precise introduction of small genomic mutations. Here we developed a system for CRISPR-based genome editing in S. suis, utilizing linear DNA fragments for homologous recombination (HR) and a plasmid-based negative selection system for bacteria not edited by HR. To enable the use of this system in other bacteria, we engineered a broad-host-range replicon in the CRISPR plasmid. We demonstrated the utility of this system to rapidly introduce multiple gene deletions in successive rounds of genome editing and to make precise nucleotide changes in essential genes. Furthermore, we characterized a mechanism by which S. suis can escape killing by a targeted Cas9-sgRNA complex in the absence of HR. A characteristic of this new mechanism is the presence of very slow-growing colonies in a persister-like state that may allow for DNA repair or the introduction of mutations, alleviating Cas9 pressure. This does not impact the utility of CRISPR-based genome editing because the escape colonies are easily distinguished from genetically edited clones due to their small colony size. Our CRISPR-based editing system is a valuable addition to the genetic toolbox for engineering of S. suis, as it accelerates the process of mutant construction and simplifies the removal of antibiotic markers between successive rounds of genome editing.
Lactobacilli are naturally found in the gastrointestinal tract of chickens, and there is interest in utilizing autochthonous
strains for the delivery of therapeutic proteins. Previously we identified three chicken-derived Lactobacillus strains, Lactobacillus agilis La3, Lactobacillus vaginalis Lv5, and Lactobacillus crispatus Lc9, which persist in the gastrointestinal tract of chickens fed either a commercial or high-protein diet. In the current
study, we investigated the ability to electrotransform these strains, determined plasmid vector stability, and compared reporter
gene expression directed by several different promoters. The La3 and Lv5 strains were reproducibly transformed with efficiencies
of 108 and 106 transformants per microgram of plasmid DNA, respectively. The third strain tested, L. crispatus Lc9, was recalcitrant to all transformation protocols examined. The plasmid vectors pTRK563 and pTRKH2 were maintained over
100 generations in La3 and Lv5, respectively. The ability of La3 and Lv5 to express the heterologous reporter gene gfp was analyzed using heterologous and homologous promoters. Transformants of both La3 and Lv5 containing the La3 ldhL promoter were the most fluorescent. To our knowledge, this is the first report of successful transformation and heterologous
protein expression in L. agilis and L. vaginalis. The ability of these strains to express heterologous proteins in vitro indicates their potential utility as in vivo delivery vectors for therapeutic peptides to the chicken gastrointestinal tract.
