转化条件对质粒DNA转化大肠杆菌的影响

研究了质粒DNA大小、质粒DNA浓度、CaCl2 浓度、热休克时间及感受态细胞保藏时间等因素对大肠杆菌HB1 0 1和JM1 0 5转化频率的影响 ,并对转化子中质粒DNA进行了分离、酶切、琼脂糖凝胶电泳检测。结果表明 ,CaCl2 浓度、质粒大小和浓度 ,以及感受态细胞的活力对转化频率有重要影响 ,42℃热休克处理可以提高转化频率。     

一种简单的哺乳动物细胞附着体质粒CaCl2转化方法

目的:建立一种简单经济的哺乳动物细胞附着体质粒还原实验方法。方法:构建附着体载体,转染中国卵巢仓鼠(CHO)细胞和小鼠脑神经瘤细胞Neuro-2a,利用改良的赫特裂解法提取附着体质粒,CaCl2法转化附着质粒至宿主菌大肠杆菌DH5α,再次从DH5α中提取质粒,将转染前后质粒用KpnⅠ/BamHⅠ双酶切和BamHⅠ单酶切,并将转染前后的质粒进行DNA测序分析。结果:与最初转染的质粒相比,还原质粒双酶切和单酶切后的条带大小一致;DNA测序分析表明,转染前后质粒中的插入序列相同。结论:建立了可用于质粒还原实验的简单的CaCl2转化方法。     

质粒pMC71A—LEU转化阴沟肠杆菌E26—1的研究

质粒pMC71A－LEU在对阴沟肠杆菌E26－1（EnterobactercloacaeE26－1）的转化中研究了感受态细胞、CaCl2浓度、热激温度和质粒DNA量对转化频率的影响。结果表明,在阴沟肠杆菌F26－1生长到OD600＝0．45时,用50mmol／LCaCl2处理细胞,加入100ng质粒DNA,在32℃热激2min,转化频率最高,可达1×103转化子／μgDNA。     

一种简单、快速制备与转化大肠杆菌受体细胞的方法

用TSB溶液取代常规的CaCl2溶液,制备大肠杆菌受体细胞。在菌体处于对数早期时离心收集细胞,用TSB缓冲液悬浮后,即可用于质粒DNA的转化。转化效率可达107～108转化子／μgDNA。     

3种假单胞菌CaCl2法感受态细胞制备及转化条件

假单胞菌因其生境和代谢类型的多样性,在污染环境修复、生物转化、生物防治等领域具有广阔的应用潜力;外源基因的导入是假单胞菌遗传改造的重要环节,而感受态细胞的制备和转化方法的建立是导入外源基因的重要方法学基础.本研究以从石油污染土壤中分离筛选的假单胞菌属的3个菌株Pseudomonas putida TS11、P.stutzeri DNB、P.mendocina JJ12为对象,通过3因素4水平正交实验设计,研究了不同CaCl2浓度、热激时间及复苏时间对不同假单胞菌感受态细胞制备及转化效率的影响.结果表明:CaCl2浓度是影响假单胞菌转化效率的最主要因素(P＜0.05),且在制备感受态细胞之前用无菌蒸馏水多次洗涤菌体细胞,转化率明显提高.3种假单胞菌的CaCl2转化优化条件分别为:100 mmol·L-1 CaCl2,热激3 min,复苏1.5 h;50 mmol·L-1 CaCl2,热激6 min,复苏1.5 h;75 mmol·L-1 CaCl2,热激4.5 min,复苏0.5h.在上述转化条件下,3种假单胞菌的外源质粒转化效率均达到105个转化子·μg-1DNA水平.     

原生质体介导马尔尼菲青霉基因转化体系的优化

目的优化原生质体介导的马尔尼菲青霉转化体系,为其基因功能研究提供良好的平台。方法通过原生质体介导将构巢曲霉(Aspergillus nidulans)pyrG基因插入马尔尼菲青霉尿嘧啶缺陷株ligD(pyrG-,ligD-)中,在不含尿嘧啶的培养基中筛选阳性转化子,运用PCR验证重组子,通过改变影响转化效率的酶解时间、培养基浓度、质粒浓度、不同配方的原生质体洗涤液STC和不同配方的原生质体助融剂PTC 5个条件对体系进行优化。结果 PCR验证A.nidulans pyrG基因成功的插入ligD中,转化子可稳定传代。最适合ligD原生质体转化效率的条件为:酶解时间6h,PTC(60%PEG-4000,100mmol/L Tris-HCl pH8.0,100mmol/L CaCl2),每100μL原生质体(106~107/mL)加入2.5μg质粒,0.6 mol/L蔗糖SD/SDU筛选/再生培养基,每1μg质粒转化子可达68个左右。结论成功优化了原生质体介导马尔尼菲青霉转化体系的条件,优化后该方法转化效率高,为基因功能研究提供良好平台。     

无机盐对阿维链霉菌接合转移及异源表达放线紫红素的影响

摘要：【目的】阿维链霉菌可作为异源表达抗生素生物合成基因簇的良好宿主,但是需要优化含有大片段DNA质粒的接合转移效率。【方法】我们选取MgCl2、NaCl、Ca(NO3)2 和CaCl2等4种无机盐,在0－200 mmol/L浓度范围内分别研究其对大质粒向阿维链霉菌接合转移的影响,再设计完全随机试验筛选最佳条件。【结果】CaCl2对阿维链霉菌接合转移有极明显的促进作用,MgCl2也有一定提高作用。通过完全随机试验筛选出最佳的CaCl2和MgCl2浓度组合,使大质粒的接合转移效率提高11倍。同时,本研究还发现阿维链霉菌异源表达放线紫红素的最适培养基,成功表达放线紫红素。【结论】特定无机盐对阿维链霉菌接合转移效率有明显提高作用,并且能促进放线紫红素在阿维链霉菌中的表达。     

大肠杆菌感受态细胞转化能力的影响因素

探讨了大肠杆菌菌株、细菌生长状态、转化溶液、抗冻剂及保存时间、质粒长度和纯度对感受态细胞转化能力的影响。结果表明,以100 mmol/L CaCl2为缓冲液,采用经活化培养的A600为0.55的TG1制备的感受态细胞,在冰上放置6h后转化,所得转化率最高,可达2×106-4×107cfu/μg DNA(pUC19)。随着质粒长度增加和纯度降低,转化率有所下降。若感受态细胞要保存备用,以15％甘油为抗冻剂优于7％DMSO,但添加抗冻剂对转化率有抑制作用。贮于甘油的感受态细胞在-70℃冻存两个月后仍有较理想的转化率。     

大肠杆菌JM109感受态形成因素分析

目的:分析大肠杆菌JM109感受态形成因素,提高转化效率。方法:采用不同生长状态、不同转化溶液、不同保存时间及热激处理时间的细菌制备感受态,分析转化效率。结果:以20mmol/L MgCl2 80mmol/L CaCl2为处理液,经活化培养OD600为0.82的菌液制备感受态细胞,4℃放置12~24h之内,42℃热激处理60s,转化效率最高,可达9.8×106~1.2×107cfu/μg DNA(pUC19)。随着质粒长度增加,转化效率下降。结论:感受态细胞形成与生长状态关系密切,金属离子、有机溶剂对感受态的形成影响显著。感受态形成过程中,细胞可能发生了一系列的生理变化。     

Alpha珠蛋白基因片段表达载体的构建

目的：构建alpha珠蛋白基因片段反义表达载体,抑制重型beta地贫患者alpha珠蛋白基因的过量表达,为重型beta地贫基因治疗打下基础。方法：采用PCR扩增alpha珠蛋白基因片段807bp,反应条件94℃ 30s,68℃ 1min,72℃ 2min,35个循环,然后将所扩增片段用Klenow大片段酶补平PCR产物末端,另外用HindⅢ酶切pLNSX,用Klenow补平后,与补平末段的PCR产物平端连接,转化用CaCl2制备的感受态细胞受体DH5α,用碱法提取所得克隆子质粒,电泳比较质粒大小,再用PCR初筛,最后用双酶切进行鉴定。结果：经过质粒大小比较,PCR初筛,双酶切鉴定,得到6个正向插入重组子。     

Studies on transformation in Shigella

Transformation of pBR322 DNA into Shigella occurred at a low frequency. The efficiency of transformation was highest in S. dysenteriae 1 and lowest in S. flexneri. Treatment of cells with CaCl2 for a prolonged period (24h) increased the efficiency of transformation in all strains, except in S. flexneri, where transformation efficiency could not be improved by a variety of manipulations. Transformation efficiency did not increase in any of the strains when transformation was carried out with plasmid DNA obtained from a transformant (homologous transformation), suggesting the absence of a strong restriction-modification system. Extracellular deoxyribonuclease (DNase) levels were low in all the strains tested, but the levels of endogenous DNAse, released after CaCl2 treatment or sonication of the cells, were high. Washing the cells with a solution of CaCl2 did not enhance transformation, suggesting that endogenous DNase could be a significant factor affecting transformation efficiency in species of Shigella.     

Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori

In this study, the efficiency of gene replacement in Aspergillus awamori between Agrobacterium-mediated transformation and CaCl(2)/PEG-mediated transformation was compared. For the genes, pyrG and gfaA, it was found that the homologous recombination frequencies obtained by Agrobacterium-mediated transformation were 3- to 6-fold higher than the frequencies obtained with CaCl(2)/PEG protoplast transformation. For the pyrG gene, it was found that Agrobacterium-mediated transformation allowed an efficient homologous recombination with shorter DNA flanks than CaCl(2)/PEG protoplast transformation. Finally, the addition of the dominant amdS marker as a second selection marker to the gene replacement cassette led to a further 2-fold enrichment in transformants with gene replacement events, resulting in a gene replacement frequency of 55%. Based on the data it can be concluded that Agrobacterium-mediated transformation is an efficient tool for gene replacement and that the amdS gene can be successfully used as a second selection marker to select transformants with putative gene replacement.     

Transformation and transfection of Pseudomonas aeruginosa: effects of metal ions.

The ability of different metal ions to promote transformation of Pseudomonas aeruginosa by deoxyribonucleic acid of the plasmid RP1 was examined. CaCl2, MgCl2, and MnCl2 were found to promote such transformation, although at different frequencies and with the optimum response at different concentrations. Only MgCl2 promoted transfection of P. aeruginosa by the linear deoxyribonucleic acid of phage F116. CaCl2 was demonstrated to allow adsorption and entry into the cell of F116 deoxyribonucleic acid such that it became resistant to exogenous deoxyribonuclease, but phage production occurred only when MgCl2 was provided. Inactivation of linear phage deoxyribonucleic acid taken up in the absence of MgCl2 was observed. The transfection frequencies at various concentrations of MgCl2 were compared, and the optimum response occurred at the concentration which promoted the highest frequency of transformation by RP1 deoxyribonucleic acid.     

Stabilization of glucocorticoid receptors in vitro using divalent cations plus cation chelators

The specific glucocorticoid receptor binding of rat liver cytosol was very unstable in vitro at 25 and 4 degrees C. However, 5 mM CaCl2 added with 5 mM EDTA to cytosol prior to incubation markedly stabilized unbound glucocorticoid receptors at both temperatures. Optimal effectiveness was achieved using equimolar (5 mM) amounts of CaCl2 and EDTA. On the other hand, 5 mM CaCl2 (added alone) further destabilized the unbound glucocorticoid receptor, while 5 mM EDTA (added alone) had no effect at 25 degrees C. EGTA (in lieu of EDTA) added with CaCl2 stabilized hepatic receptor binding at 25 degrees C. On the other hand, citrate added with calcium was ineffective in stabilizing the hepatic glucocorticoid receptor. MgCl2 effectively replaced CaCl2 as a stabilizing agent at 25 degrees C if added with 5 mM EDTA. When added alone, MgCl2 slightly destabilized the unbound receptor. Sucrose density gradient analysis (in low salt) revealed that CaCl2 plus EDTA enhanced the steroid-receptor complex sedimentation coefficient from 7 S to about 10 S. Unlike molybdate, CaCl2 plus EDTA had no apparent effect on steroid-receptor complex thermal transformation into a nuclear binding form, while MgCl2 plus EDTA partially reduced transformation. These results suggest a novel means to chemically stabilize unbound hepatic glucocorticoid receptors in vitro which may be of particular importance for receptor purification studies.     

Induction of Escherichia coli K-12 mutants with an increased efficiency of plasmid transformation

A new rapid method for plasmid transformation of Escherichia coli K-12 cells has been devised. It consists in application of plasmid pMB9 DNA to the surface of an agar medium with 0.05 M CaCl2 and tetracycline (50 micrograms/ml). The recipient cells treated with nitrosoguanidine were drifted on by sectors on the plates with pMB9 DNA. The method enabled the obtaining of 12 mutants with high efficiency of plasmid transformation.     

3种假单胞菌CaCl2法感受态细胞制备及转化条件

假单胞菌因其生境和代谢类型的多样性,在污染环境修复、生物转化、生物防治等领域具有广阔的应用潜力;外源基因的导入是假单胞菌遗传改造的重要环节,而感受态细胞的制备和转化方法的建立是导入外源基因的重要方法学基础.本研究以从石油污染土壤中分离筛选的假单胞菌属的3个菌株Pseudomonas putida TS11、P. stutzeri DNB、P. mendocina JJ12为对象,通过3因素4水平正交实验设计,研究了不同CaCl₂浓度、热激时间及复苏时间对不同假单胞菌感受态细胞制备及转化效率的影响.结果表明： CaCl₂浓度是影响假单胞菌转化效率的最主要因素(P<0.05),且在制备感受态细胞之前用无菌蒸馏水多次洗涤菌体细胞,转化率明显提高.3种假单胞菌的CaCl₂转化优化条件分别为：100 mmol·L^-1 CaCl₂,热激3 min,复苏1.5 h;50 mmol·L^-1 CaCl₂,热激6 min,复苏1.5 h;75 mmol·L^-1 CaCl₂,热激4.5 min,复苏0.5 h.在上述转化条件下,3种假单胞菌的外源质粒转化效率均达到10.5个转化子·μg^-1 DNA水平.     

A rapid and efficient method for plasmid transformation of Klebsiella pneumoniae and Escherichia coli

A rapid and efficient method for plasmid transformation of Klebsiella pneumoniae M5a1 and Escherichia coli K12 has been developed. The method, which uses a freeze-thaw cycle in the presence of CaCl2 to facilitate DNA uptake, is substantially more efficient for K. pneumoniae M5a1 than the conventional transformation procedure for E. coli. The simplicity and speed of the method makes it very attractive for routine transformation of K. pneumoniae M5a1 and E. coli K12.     

Efficient transformation of Serratia marcescens with pBR322 plasmid DNA

Eight Serratia marcescens strains tested could be transformed with the plasmid pBR322. Transformants were selected on the basis of resistance to high levels of ampicillin (400 to 500 micrograms/ml). For six of the strains, the CaCl2- mediated transformation procedure developed for Escherichia coli was successful. For the other two strains, no transformants were obtained with the CaCl2-mediated transformation procedure unless the cells first received a heat treatment. Transformation frequency was dependent on DNA concentration, and no transformation was detected with linear pBR322 DNA. The stability and copy number of pBR322 were similar in S. marcescens and E. coli. As in E. coli, the pBR322 DNA was amplified in S. marcescens after inhibition of proteins synthesis. Based on these results, cloning in S. marcescens should be possible and pBR322 should be a useful cloning vehicle.     

Ca2+ -dependence and inhibition of transformation by trifluoperazine and chlorpromazine in Thermoactinomyces vulgaris

Ca(2+) enhanced the transformation frequency of Thermoactinomyces vulgaris (stock no. 1278) of an auxotrophic strain by the chromosomal DNA isolated from a prototrophic strain (stock no. 1227). The number of transformants showed a marked increase with increasing concentration of CaCl(2) upto 0.05 mM; and above this concentration, the transformation frequency decreased significantly. Antipsychotic drugs that are potent calmodulin inhibitors, like trifluoperazine and chlorpromazine, when applied in the concentration range of 0.01-0.04 mM along with optimal CaCl(2) concentration to the cultures of the recipient cells, resulted in a significant inhibition in the frequency of Ca(2+)-stimulated transformation. The results of present investigation suggest the involvement of a Ca(2+)-dependent protein activator in the development of Ca(2+)-mediated competence, which could have played an important role in the enhancement of genetic transformation in this aerobic spore forming thermophilic actinomycete.