Background:The evolution of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) has been accelerated recently by the indiscriminate application of antibiotics. Antibiotic resistance has challenged the success of medical interventions and therefore is considered a hazardous threat to human health.Objectives:The present study aimed to describe the use of zinc finger nuclease (ZFN) technology to target and disrupt a plasmid-encoded β-lactamase, which prevents horizontal gene transfer-mediated evolution of ARBs.Materials and Methods:An engineered ZFN was designed to target a specific sequence in the ampicillin resistance gene (ampR) of the pTZ57R plasmid. The Escherichia coli bacteria already contained the pZFN kanamycin-resistant (kanaR) plasmid as the case or the pP15A, kanaR empty vector as the control, were transformed with the pTZ57R; the ability of the designed ZFN to disrupt the β-lactamase gene was evaluated with the subsequent disturbed ability of the bacteria to grow on ampicillin (amp) and ampicillin-kanamycin (amp-kana)-containing media. The effect of mild hypothermia on the ZFN gene targeting efficiency was also evaluated.Results:The growth of bacteria in the case group on the amp and amp-kana-containing media was significantly lower compared with the control group at 37°C (P < 0.001). Despite being more efficient in hypothermic conditions at 30°C (P < 0.001), there were no significant associations between the incubation temperature and the ZFN gene targeting efficiency.Conclusions:Our findings revealed that the ZFN technology could be employed to overcome ampicillin resistance by the targeted disruption of the ampicillin resistance gene, which leads to inactivation of β-lactam synthesis. Therefore, ZFN technology could be engaged to decrease the antibiotic resistance issue with the construction of a ZFN archive against different ARGs. To tackle the resistance issue at the environmental level, recombinant phages expressing ZFNs against different ARGs could be constructed and released into both hospital and urban wastewater systems.

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is still the common host for ing and heterologous protein expression. Various strategies have been employed to increase protein expression in , but, it seems that external factors such as selection marker concentration can drastically affect the yield of protein and plasmid. Alterations of protein expression and plasmid yields of in different concentrations of ampicillin, as selection marker, will be determined. In order to improve heterologous expression, the system will be redesigned and optimized. The expression cassette of codon optimized EGFP for was synthesized in pUC57. The pUC57-GFP was transformed into. The expression of GFP was verified by SDS-PAGE and flow cytometry after induction by IPTG (0.5 mM) and incubation with 0, 100, 200 and 300 μg.mL ampicillin. Plasmid copy numbers of samples were determined by Real-Time PCR on AMP gene using regression line of diluted standard curve. GFP expressing clones formed fair green colonies on LB agar supplemented with 0.5 mM IPTG and showed fluorescence in FL1 filter of flow cytometry and an extra protein band on SDS-PAGE gel. The fluorescent intensity of GFP in 0, 100, 200 and 300 μg.mL ampicillin in medium were 549.83, 549.78, 1443.52, 684.87, and plasmid copy numbers were 6.07×10 , 3.21×10 , 2.32×10 , 8.11×10 , respectively. The plasmid yields were 55 ng.μL, 69 ng.μL, 164 ng.μL and 41 ng.μL, respectively. Protein and plasmid yields of are variable in different concentrations of ampicillin and need to be optimized in newly designed expression systems. Protein and plasmid yield in the optimized concentration (200 μg.mL) was significantly (p < 0.01) higher than other doses.

Elevation of hemoglobin F (HbF) ameliorates symptoms of β‐thalassemia, as a common autosomal recessive disorder. In this study, the ability of an engineered zinc‐finger nuclease (ZFN) system was assesed to disrupt the KLF1 gene to inhibit the γ to β hemoglobin switching in K562 cells. This study was performed using a second generation integration‐deficient lentiviral vector assigned to transient gene targeting. The sequences coding for zinc finger protein arrays were designed and subcloned in TDH plus as a transfer vector. Transduction of K562 cells was performed with the integrase minus lentivirus containing ZFN. The indel percentage of the transducted cells with lentivirus containing ZFN was about 29%. Differentiation of K562 cell line into erythroid cell lineage was induced with cisplatin concentration of 15 µg/mL. After differentiation, γ‐globin and HbF expression were evaluated using real‐time reverse‐transcription polymerase chain reaction and hemoglobin electrophoresis methods. The levels of γ‐globin messenger RNA were nine‐fold higher in the ZFN treated cells compared with untreated cells 5 days after differentiation. Hemoglobin electrophoresis method showed the same results for HbF level measurement. Application of the ZFN tool to induce KLF1 gene mutation in adult erythroid progenitors might be a candidate to stimulate HbF expression in β‐thalassemia patients.

Background: Nanotechnology has introduced valuable carriers for vaccine delivery. The success of vaccination depends on many factors, such as the intact and safe presentation of vaccine candidates to immune cells. We have conjugated branched PEI-2k and oleic acid (OL) as the building block of the cationic micelle. We aimed to introduce a novel carrier for vaccine candidates. Materials and Methods: We conjugated polyethyleneimine and OL (POA) to synthesize the building blocks of cationic micelles. The critical micelle concentration (CMC), size and zeta potential of micelles, and their stability in 60 days were determined. Loading, encapsulation efficiency, and in vitro release study were assessed using bovine serum albumin (BSA) as a protein model. Furthermore, the cytotoxicity and hemocompatibility of developed nanosized micelles were evaluated to ascertain the biocompatibility of fabricated micelles. Cell uptake of cationic micelles in the macrophage cell line was also followed up. Results: The conjugation of two polymer parts was confirmed by Fourier transform infrared spectroscopy and 1 H nuclear magnetic resonance techniques. The CMC of the developed micelles was around 5.62 × 10 −8 mg / ml, whereas the loading and encapsulation efficiencies were 16.5% and 70%, respectively. The size and zeta potential of the cationic micelles were 96.53 ± 18.53 nm and 68.3 mV, respectively. The release of BSA from POA micelles after 8 and 72 hours was 8.5% and 82%, respectively. Finally, fluorescence microscopy showed that the prepared micelles were successfully and effectively taken up by RAW264.7 cells. Conclusion: These results may provide a cutting-edge vaccine delivery solution and open up a new avenue for future vaccine research.