Plasmid DNA Analysis of Pathogenic Escherichia coli in Musk Deer

[Objective] The pathogenic Escherichia coli in musk deer was classified at molecular level to provide basic materials for molecular epidemiology of pathogenic Escherichia coli in musk deer. [Method] Plasmids from 24 pathogenic Escherichia coli in musk deer were extracted by the Lysis Triton method, and then identified by single enzyme digestion with three endonucleases of Hind Ⅲ, EcoR Ⅰ and BamH Ⅰ. [Result] The yield rate of plasmids was 91.6%, and 24 pathogenic Escherichia coli in musk deer had the identical or similar plasmid profiles. [Conclusion] Plasmid DNA analysis offers scientific basis for molecular epidemiology of pathogenic Escherichia coli in musk deer in Sichuan Institute of Musk Deer Breeding.

Catalytic hydrolysis of phosphate diester (BNPP) and plasmid DNA by mononuclear macrocyclic polyamine metal complexes

The activities of the catalytic hydrolysis of phosphate diester （BNPP） [bis（p-nitrophenyl）phosphate diester] and plasmid DNA （pUC 18） by mononuclear macrocyclic polyamine metal complexes have been investigated in this paper. The results showed that the highest activity in hydrolysis of BNPP was obtained with le--Zn（II） complex （composed of lipophilic group） as catalyst. The hydrolysis rate enhancement is up to 3.64 × 10^4 fold. These metal complexes could effectively promote the cleavage of plasmid DNA （pUC18） at physiological conditions.

Effects of medium composition on the production of plasmid DNA vector potentially for human gene therapy

Plasmid vector is increasingly applied to gene therapy or gene vaccine. The production of plasmid pCMV-AP3 for cancer gene therapy was conducted in a modified MBL medium using a recombinant E. coli BL21 system. The effects of different MMBL components on plasmid yield, cell mass and specific plasmid DNA productivity were evaluated on shake-flask scale. The results showed that glucose was the optimal carbon source. High plasmid yield (58.3 mg/L) was obtained when 5.0 g/L glucose was added to MMBL. Glycerol could be chosen as a complementary carbon source because of the highest specific plasmid pro- ductivity (37.9 mg DNA/g DCW). After tests of different levels of nitrogen source and inorganic phosphate, a modified MMBL medium was formulated for optimal plasmid production. Further study showed that the initial acetate addition (less than 4.0 g/L) in MMBL improved plasmid production significantly, although it inhibited cell growth. The results will be useful for large-scale plasmid production using recombinant E. coli system.

Optimization on cationic liposome-mediated cell transfection of plasmid DNA

Objective:The development of gene carriers for efficient gene delivery into cells has attracted growing attention in recent years.The aim of this study was to achieve a better outcome of AAV-293 cells transfection by plasmid DNA.Methods:We studied the optimal condition for higher efficiency of cationic lipid-mediated cell transfection.Four experimental groups were set.Plasmid DNA and liposome were mixed in each groups at different ratios(μg:μL),1:2.5,1:3.5,1:4.0 and 1:5.0,respectively.LacZ gene functioned as reporter gene,measuring the transfection efficiency of the four groups using the method of X-gal staining.Results:When the ratio was 1:3.5,the cell transfection rate was the highest.While the ratio of 1:2.5 recommended by product manual achieve the lowest transfection rate.Their difference had statistical significance.Conclusion:In order to obtain a higher transfection efficiency,optimization on conditions of the ratio of plasmid DNA to liposome is necessary in cell transfection.

Amplification of plasmid DNA bound on soil colloidal particles and clay minerals by the polymerase chain reaction

Polymerase chain reaction （PCR） was used to amplify a 600-base pair （bp） sequence of plasmid pGEX-2T DNA bound on soil colloidal particles from Brown soil （Alfisol） and Red soil （Ultisol）, and three different minerals （goethite, kaolinite, montmorillonite）. DNA bound on soil colloids, kaolinite, and montmorillonite was not amplified when the complexes were used directly but amplification occurred when the soil colloid or kaolinite-DNA complex was diluted, 10- and 20-fold. The montmorillonite-DNA complex required at least 100-fold dilution before amplification could be detected. DNA bound on goethite was amplified irrespective of whether the complex was used directly, or diluted 10- and 20-fold. The amplification of mineral-bound plasmid DNA by PCR is, therefore, markedly influenced by the type and concentration of minerals used. This information is of fundamental importance to soil molecular microbial ecology with particular reference to monitoring the fate of genetically engineered microorganisms and their recombinant DNA in soil environments.

Preparation of Ag/AgBr/TiO_2 as Catalyst Carriers and Its Damage to Plasmid DNA and Tetrahymena

The composites based on the Ti O2 are potentially used in wetland pollution control. In this work, the biological effect of the Ag/Ag Br/Ti O2/Active carbon(AC) composites was studied on the plasmid DNA and Tetrahymena membrane. The atomic force micrograph(AFM) images showed that, in the presence of the composites under illumination, most p UC18 DNA molecules showed quite different topography and were opened and relaxed circle shapes. After DNA was catalyzed for 40 min, all supercoiled and circular DNA were changed into the linear DNA molecules. The gel electrophoresis experiment confirmed the results and demonstrated the dynamic process of DNA degradation. ATR-FTIR spectra revealed that amide groups and PO2-of the phospho-lipid phospho-diester on Tetrahymena surface were oxidized in the presence of the composites under illumination. An increase in the fluorescence polarization of DPH was observed, reflecting a significant decrease in membrane fluidity of Tetrahymena.

Delivery of Plasmid DNA into Tumors by Intravenous Injection of PEGylated Cationic Lipoplexes into Tumor-Bearing Mice

For systemic injection of cationic liposome/plasmid DNA (pDNA) complexes (cationic lipoplexes), polyethylene glycol (PEG)-modification (PEGylation) of lipoplexes can enhance their systemic stability. In this study, we examined whether intravenous injection of PEGylated cationic lipoplexes into tumor-bearing mice could deliver pDNA into tumor tissues and induce transgene expression. PEGylation of cationic liposomes could prevent their agglutination with erythrocytes. However, when PEGylated cationic lipoplexes were injected intravenously into tumor-bearing mice, they accumulated in tumor vascular vessels and did not exhibit transgene expression in tumors with both poor and well-developed vascularization. Furthermore, PEGylated cationic lipoplexes of CpG- free pDNA could not increase transgene expression in tumors after intravenous injection. These results suggested that PEGylation could not extravasate cationic lipoplexes from vascular vessels in tumors and abolished transgene expression although it enhanced the systemic stability of cationic lipoplexes by avoiding interactions with blood components such as erythrocytes. Successful delivery of pDNA to tumors by PEGylated cationic liposomes will require a rational strategy and the design of liposomal delivery systems to overcome the issue associated with the use of PEG.

Destruction of <i>Escherichia coli</i>and Broad-Host-Range Plasmid DNA in Treated Wastewater by Dissolved Ozone Disinfection under Laboratory and Field Conditions

Broad-host-range plasmids are frequently associated with antibiotic resistance genes and can quickly spread antibiotic resistant phenotypes among diverse bacterial populations. Wastewater treatment plants have been identified as reservoirs for broad-host-range plasmids carrying resistance genes. The threat of broad-host-range plasmids released into the environment from wastewater treatment plants has identified the need for disinfection protocols to target broad-host- range plasmid destruction. Here we evaluate the efficacy of dissolved ozone at 2 and 8 mg·L–1 as a primary means for the destruction of broad-host-range plasmid and chromosomal DNA in simulated effluent. Pilot-scale tests using an experimental unit were carried out in municipal wastewater treatment plant effluent and compared with ultraviolet (UV)-irradiation and chlorination methodologies. Genes specific to Escherichia coli (uidA) and IncP broad-host-range plasmids (trfA) were monitored using real-time quantitative polymerase chain reaction (qPCR), and total DNA was monitored using absorbance spectroscopy. In wastewater treatment plant experiments, E. coli qPCR results were compared to a recognized culture-based method (Colilert?) for E. coli. In laboratory experiments, dissolved ozone at 8 mg·L–1 significantly destroyed 93% total, 98% E. coli, and 99% of broad-host-range plasmid DNA. Ozonation, UV-irradiation, and chlorination significantly reduced DNA concentrations and culturable E. coli in wastewater treat- ment plant effluent. Chlorination and UV disinfection resulted in 3-log decreases in culture-based E. coli concentrations in wastewater treatment plant effluent while changes were not significant when measured with qPCR. Only ozonation significantly decreased the IncP broad-host-range plasmid trfA gene, although concentrations of 2.2 × 105 copies trfA·L–1 remained in effluent. Disinfection processes utilizing high dissolved ozone concentrations for the destruction of emerging contaminants such as broad-host-range plasmid and total DNA may have utility as methods to ensure downstream environmental health and safe water reuse become more important.

Uhrf1基因重组腺病毒载体构建及其在小鼠心肌细胞DNA损伤修复中的作用研究

目的:构建携带小鼠泛素样同源域和环指结构域1(Uhrf1)基因的重组腺病毒载体,验证Uhrf1基因在原代乳鼠心肌细胞中的表达情况,并探究其在过氧化氢(H_(2)O2)诱导的心肌细胞DNA损伤中的作用。方法:利用PCR扩增小鼠Uhrf1基因的编码序列,将其酶切后插入pADM-CMV-C-FH载体,获得重组腺病毒质粒ADM-Uhrf1。将该质粒转染至HEK293T细胞包装成重组腺病毒颗粒,数代扩增后进行腺病毒的纯化及滴度检测。分离25只1日龄ICR小鼠原代心肌细胞,分为两组,以感染复数(MOI)为50的比例分别感染ADM-Uhrf1及ADM-control(ADMCtrl),通过Western blot及免疫荧光染色验证重组腺病毒介导的UHRF1蛋白的表达,并利用H_(2)O2诱导心肌细胞DNA损伤,进而探究Uhrf1在DNA损伤修复过程中的作用。结果:通过壳蛋白免疫法检测得到的ADM-Uhrf1病毒滴度为1.8×10^(13) pfu/L。Western blot验证显示UHRF1蛋白表达水平显著升高(P<0.05),免疫荧光染色显示UHRF1主要表达在细胞核内,且Uhrf1的过表达能够显著抑制DNA损伤标志物磷酸化组蛋白H_(2)A变异体(γH_(2)AX)蛋白的表达(P<0.01)。结论:成功构建了携带小鼠Uhrf1基因的重组过表达腺病毒载体,并通过腺病毒递送系统在心肌细胞中实现了Uhrf1的过表达,且Uhrf1的过表达有效减轻了H_(2)O2诱导的心肌细胞DNA损伤。

质粒DNA Fast NGS测序方法的开发和应用

质粒DNA是最常用的基因运载工具,在基因合成技术中扮演着至关重要的角色。如何实现合成质粒DNA的准确且快速检测,是确保基因组完整性和提高基因合成效率的关键。尽管基于一代DNA的测序方法,其准确性已成为行业标准,但在检测通量、检测速度和检测成本等方面仍然存在局限性,这促使科学家们不断寻求新的解决方案。基于生物酶库,开发了DNA建库酶TN5,建立了高通量质粒DNA检测方案——Fast NGS。利用不同长度、不同质量的质粒DNA样本评估了Fast NGS的可行性,并对质粒DNA样本进行了高通量测序,最后对比了Fast NGS与Sanger测序的效率。结果表明,DNA建库酶TN5蛋白的纯度和质量符合二代测序要求。Fast NGS适用于3~8 kb基因合成质粒的测序检测,其检测通量高达2500个·12 h-1,测序成功率超过95%,测序准确性与一代测序相当,并且无明显序列偏好性。Fast NGS实现了质粒DNA的高通量、快速且低成本检测,为基因合成技术的发展提供了新的方向。

指数补料联合升温发酵法提高治疗性HPV DNA疫苗产量的研究进展

宫颈癌是全球女性第四大常见癌症。目前上市的HPV疫苗均为预防性疫苗,对已感染HPV人群无治疗效果。HPV感染率仍然很高。近年来,科学家日益关注治疗性HPV疫苗的研发,可诱导细胞免疫杀死已感染HPV的细胞。治疗性HPV DNA疫苗以其低成本、稳定性好的特点备受青睐。为降低治疗性HPV DNA疫苗的成本,目前多采用大肠杆菌高密度发酵生产。本研究使用具有热敏感特性的DNA质粒特性的治疗性HPV DNA疫苗,采用摇瓶培养载有DNA质粒的大肠杆菌,30℃培养一定时间后,分别升温至37℃或42℃,结果显示,升温至42℃组比升温至37℃组的质粒产量明显升高;采用发酵方式培养大肠杆菌,30℃培养一定时间后,升温至42℃进一步提高质粒的拷贝数;分别探究恒速补料和指数补料两种方式联合升温发酵策略对DNA质粒产量的影响,结果显示,相较于恒速补料,指数补料可显著提高升温发酵过程中DNA质粒的产量,从125.76 mg·L^(-1)提升至619.94 mg·L^(-1)。发酵得到的治疗性HPV DNA疫苗可在293T细胞中高效表达,并能在体内引起HPV抗原特异性细胞免疫反应。本研究认为指数补料联合升温发酵策略对其他DNA质粒的发酵也有一定的借鉴意义,也为CAR-T等细胞产品、RNA相关产品提供高质量DNA质粒提供参考。

一种新型的石墨烯量子点在光断裂DNA方面的应用

研究发现及验证了一种新型的石墨烯量子点(GQDs),该量子点具有高效产生单线态氧的能力,并在光断裂DNA领域展现出显著的应用潜力。在可见光照射下,无论是在有氧条件还是厌氧条件下,这种GQDs都能有效地切割质粒pBR322 DNA。通过DNA凝胶电泳方法,证实了GQDs在光照射下产生单线态氧的能力,并定量评估了其对DNA的切割效果。在有氧和厌氧条件下,GQDs均显示出优异的DNA切割活性,这一结果为深刻理解GQDs与DNA的相互作用机制提供了新的视角。

In Vitro Antioxidant and Radio Protective Activities of Lycopene from Tomato Extract against Radiation—Induced DNA Aberration

Background: The accumulation of free radicals is linked to a number of diseases. Free radicals can be scavenged by antioxidants and reduce their harmful effects. It is therefore essential to look for naturally occurring antioxidants that come from plants, as synthetic antioxidants are toxic, carcinogenic and problematic for the environment. Lycopene is one of the carotenoids, a pigment that dissolves in fat and has antioxidant properties. Materials and Methods: The antioxidant and free radical scavenging activity were assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The impact of lycopene on bacteria (E. coli) susceptibility to γ-radiation was examined by radio sensitivity assay. The study also examined the induction of strand breaks in plasmid pUC19 DNA and how lycopene extract protected the DNA from γ-radiation in vitro. Results: At varying concentrations, lycopene demonstrated its ability to scavenge free radicals such as 2, 2-diphenyl-1-picrylhydrazyl (DPPH). IC50 for lycopene was determined at 112 μg/mL which was almost partial to IC50 of standard antioxidant L-ascorbic acid. The D10 value 180 Gy of E. coli was found to be >2-fold higher in the extract-containing lycopene sample than in the extract-free controls. The lycopene extracts inhibited the radiation-induced deterioration of the plasmid pUC19 DNA. At an IC50 concentration, lycopene provided the highest level of protection. Conclusion: Lycopene functions as an efficient free radical scavenger and possible natural antioxidant source. For cancer patients and others who frequently expose themselves to radiation, lycopene may be a useful plant-based pharmaceutical product for treating a variety of diseases caused by free radicals.

Functionalization of Single- and Multi-Walled Carbon Nanotubes with Cationic Amphiphiles for Plasmid DNA Complexation and Transfection

The possibility of delivering DNA effi ciently to cells represents a crucial issue for the treatment of both genetic and acquired diseases.However,even although the effi ciency of non-viral transfection systems has improved in the last decade,none have yet proven to be suffi ciently effective in vivo.We report herein our results on the functionalization of single-walled carbon nanotubes(SWNT)and multi-walled carbon nanotubes(MWNT)by two cationic amphiphiles(lipid RPR120535 and pyrenyl polyamine),their use for the complexation of plasmid DNA,and their efficiency in transfecting cells in vitro.The experiments have shown that the efficiency of transfection is higher when using SWNT instead of MWNT,and that transfection effi ciency is similar or slightly higher when using nanoplexes(SWNT/lipid RPR120535/DNA)instead of lipoplexes(lipid RPR120535/DNA)and several orders of magnitude higher than that of naked DNA.This study therefore shows both that the transfection is better when using SWNTs and that it is dependent on the nature of the amphiphilic molecules adsorbed on the nanotubes.

Plasmid DNA induces dodecyl triethyl ammonium bromide to aggregate into vesicle

Single-chained cationic surfactant dodecyl triethyl ammonium bromide and plasmid DNA together can form vesicles once the concentration of plasmid DNA reaches a critical value （Cove）. Bigger the size of plasmid DNA, higher the value of Cove.

Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads(SSMBs)

Plasmid DNA(pDNA)isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research.Almost all pDNA purification in-volves disruption of bacteria,removal of membrane lipids,proteins and genomic DNA,purifi-cation of pDNA from bulk lysate,and concentration of pDNA for downstream applications.While many liquid-phase and solid-phase pDNA purification methods are used,the final pDNA preparations are usually contaminated with varied degrees of host RNA,which cannot be completely digested by RNase A.To develop a simple,cost-effective,and yet effective method for RNA depletion,we investigated whether commercially available size selection magnetic beads(SSMBs),such as Mag-Bind®TotalPure NGS Kit(or Mag-Bind),can completely deplete bacterial RNA in pDNA preparations.In this proof-of-principle study,we demonstrated that,compared with RNase A digestion and two commercial plasmid affinity purification kits,the SSMB method was highly efficient in depleting contaminating RNA from pDNA minipreps.Gene transfection and bacterial colony formation assays revealed that pDNA purified from SSMB method had superior quality and integrity to pDNA samples cleaned up by RNase A digestion and/or commercial plasmid purification kits.We further demonstrated that the SSMB method completely depleted contaminating RNA in large-scale pDNA samples.Furthermore,the Mag-bind-based SSMB method costs only 5-10%of most commercial plasmid purification kits on a per sample basis.Thus,the reported SSMB method can be a valuable and inexpensive tool for the removal of bacterial RNA for routine pDNA preparations.

质粒DNA实验室规模化制备工艺

目的 探索实验室规模生产质粒DNA(plasmid DNA,pDNA)的制备工艺。方法 选用质粒pEGFP-N1E. coli Stbl3菌株,用最陡爬坡试验(plackett-burman,PB)筛选出影响质粒产量最显著因素,响应面法优化重组菌高产发酵条件。采用碱裂解,浓缩质粒,通过凝胶、亲和、离子等层析分离纯化pDNA,并对所纯化的pDNA进行质量评价。结果 用PB试验和响应面试验设计筛选出的关键因素是:酵母提取物20 g/L,甘油6 g/L,接种物浓度吸光度(optical density,OD)=0.014。在最佳条件下进行3次平行发酵,生物量OD 600达28.07±2.01,质粒产量(21.34±1.31)mg/L。pDNA纯度(A260 nm/A280 nm)为1.91±0.02。内毒素含量小于0.005 EU/g DNA;几乎检测不到蛋白质及细菌基因组DNA残留,达到相关质量标准。结论 本研究采用的制备工艺可生产出高质量的p DNA。

The dynamic interactions between chemotherapy drugs and plasmid DNA investigated by atomic force microscopy

The advent of atomic force microscopy （AFM） provides a powerful tool for imaging individual DNA molecules. Chemotherapy drugs are often related to DNAs. Though many specific drug-DNA interactions have been observed by AFM, knowledge about the dynamic interactions between chemotherapy drugs and plasmid DNAs is still scarce. In this work, AFM was applied to investigate the nanoscale interactions between plasmid DNAs and two commercial chemotherapy drugs （methotrexate and cisplatin）. Plasmid DNAs were immobilized on mica which was coated by silanes in advance. AFM imaging distinctly revealed the dynamic changes of single plasmid DNAs after the stimulation of methotrexate and cisplatin. Geometric features of plasmid DNAs were extracted from AFM images and the statistical results showed that the geometric features of plasmid DNAs changed significantly after the stimulation of drugs. This research provides a novel idea to study the actions of chemotherapy drugs against plasmid DNAs at the single-molecule level.

混合模式层析分离纯化超螺旋质粒DNA

针对细胞裂解液中的超螺旋质粒DNA(sc pDNA)的分离,以质粒pVAX1为典型对象、采用Capto PlasmidSelect作为混合模式层析介质,探讨了料液中主要成分sc pDNA、开环质粒DNA(oc pDNA)和RNA的吸附行为,优化了分离条件,实现了从成分较为复杂的料液中高效分离sc pDNA。考察了上述3种组分的静态吸附,发现在(NH_(4))_(2)SO_(4)浓度c(NH_(4))_(2)SO4为1.9~2.5 mol·L^(-1)时,sc pDNA均具有较高的吸附量,确定c(NH_(4))_(2)SO_(4)=2.5 mol·L^(-1)的料液可直接上样,此时sc pDNA饱和吸附量为每克介质吸附3.3 mg。动态吸附实验发现,sc pDNA穿透略晚于oc pDNA,sc pDNA动态载量为每毫升介质负载2.00 mg,RNA吸附能力明显强于pDNA。进一步优化了洗脱、冲洗和上样量等分离条件,采用c(NH_(4))_(2)SO_(4)=2.5 mol·L^(-1)上样、c(NH_(4))_(2)SO_(4)=1.9 mol·L^(-1)冲洗、(c(NH_(4))_(2)SO_(4)=1.7 mol·L^(-1))+(cNaCl=0.3 mol·L^(-1))洗脱,sc pDNA纯度可达83.9%、同质性高达95.8%、收率为80.6%。结果表明,混合模式层析对sc pDNA选择性好、处理量较大,具有良好的应用价值。

Extraction of plasmid-like DNA and high-quality total DNA from Porphyra yezoensis

Somatic cells were prepared from sea snail enzyme digests of Porphyra yezoensis thalli. Us ing SDS - Proteinase K as extraction solution, total DNA was isolated from the somatic cells. The crude extracts of total DNA were purified with glassmilk, and the resulting DNA was of sufficient quality for digestion of restriction endonuclease. DNA bands were clearly observed in the restriction patterns of EcoRI, PstI and HaeIII respectively. The presence of DNA hands in the restriction pattern of total DNA indicated that the genome of Porphyra yezoensis may be small. Unexpectedly, using guanidinium isoth iocyanate and sarcosyl as extraction solution, a plasmid-like DNA band (2.3 Kb) was directly found in the isolated total DNA of Porphyra yezoensis. A very simple and convenient method for plasmid-like DNA isolation has been established.