用多寄主质粒pSUP1O6转化荧光假单胞菌Pfx-18

构建了荧光假单胞菌工程菌的转化系统,探索了多寄主质粒pSUP 106在不同CaCl_2浓度、不同热激时间和荧光假单胞菌受体在不同生长期的转化频率,建立了一个简便可行的荧光假单胞菌转化方法.结果表明,在荧光假单胞菌Pfx-18生长到0 D_(600)≈0.55时,用25mmol/L CaCl_2处理细胞,热激2min,转化频率最高,可达10~5/ng DNA.同时讨论了Mn~(2+)和Mg~(2+)对转化频率的影响,以及电转化的效果,为进一步利用该转化系统进行异源基因的克隆与表达研究奠定了基础.     

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.     

铜绿假单胞菌最佳电转化条件的研究

以临床分离的一株铜绿假单胞菌 (Pseudomonasaeruginosa)PA68作受体菌 ,将具有卡那霉素抗性标记的质粒pSMC2 8通过电转化导入到受体菌中 ,研究细胞生长状态、电击电压、细胞浓度、感受态细胞的贮备方式对转化效率的影响。结果表明 ,在细胞生长至OD5 40 =0 7～ 0 8时收集菌体 ,在低温 (2℃ )条件下 ,制备浓度为 10 11个细胞 mL的感受态细胞 ,在较高的电压 (2 6kV)电击下 ,能获得较高的转化效率。最高可达 1 68× 10 8个转化子 μgDNA(CFU μgDNA)。用此优化的转化条件 ,在国际上首次成功地将Mu转座复合物导入到P .aeruginosa中 ,并获得 2 4× 10 4 CFU μgDNA的高转化效率。由于Mu转座重组技术具有随机单点插入的优点 ,克服了传统转座子能在染色体上迁移的缺点 ,保证了表型的改变与转座子插入位点所在的基因突变的一一对应关系 ,为进一步研究P .aerugi nosa的基因组功能奠定基础     

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.     

嗜热厌氧乙醇菌JW200转化条件的研究

摘要 嗜热厌氧乙醇菌遗传转化系统的缺少,制约了对该菌理论基础和应用领域的进一步研究。利用聚乙二醇（PEG6000）转化和电转化技术国际首次实现了嗜热厌氧乙醇菌JW200外源基因的导入。PEG转化效率很低,因此选择对电转化条件进行优化,转化效率从4±3.2个转化子/μg质粒DNA提高到50±7.4个转化子/μg质粒DNA。实验表明获得较高的转化效率的必要条件是在细胞密度为OD660 0.2时添加甘氨酸与蔗糖后继续培养2h以及细胞在电击前的收集与洗涤保持低温。本研究为利用基因工程手段改造嗜热厌氧乙醇菌和从分子水平研究胞内乙醇代谢途径奠定了基础。     

电转化条件对大肠杆菌XL1-Blue菌株转化效率的影响

探讨XL1-Blue菌株电转化的最优条件。通过改变电压、质粒DNA浓度、细菌生长周期等影响电转化的重要条件,做出转化率的变化曲线,从中探索电转化的最优条件。实验结果得出在电容25μF、电阻200 Ω、电压2.5 kV、D600nm为0.3～0.4、0.2 cm电转化杯、DNA终浓度0.1μg/ml、感受态细胞终浓度2.5×1012、氨苄青霉素浓度50μg/ml的条件下,电转化效率最高,可达到7.64×108。电转化实验转化效率高,重复性好,为成功的建立抗体库提供了保证。     

制备高效大肠杆菌电转化感受态细胞和电转化条件的研究

旨在建立一种高效大肠杆菌电转化感受态细胞的制备方法,研究了摇瓶装液量,菌体生长阶段,转化电场强度,转化后复苏时间以及感受态细胞存放时间等对转化效率的影响.结果表明,基液量为400 mL/2L,菌体在OD600值为0.452时收集所制备的感受态细胞,在电场强度12.5 kV/cm条件下电击5 ms,转化后复苏时间2h,转化效率可达到1010CFU/μg DNA.此条件下制备的感受态细胞转化效率高,质量稳定,重复性好.     

高渗提高凝结芽孢杆菌P4-102B菌株的电击转化效率

【目的】凝结芽孢杆菌(Bacillus coagulans)是一种非常有工业应用前景的微生物,但遗传转化的困难是限制对其进行代谢工程改造的关键因素。本研究主要考察生长培养基、电击缓冲液、复苏培养基中添加高渗剂如山梨醇、甘露醇等对凝结芽孢杆菌转化效率稳定性的影响,并对凝结芽孢杆菌电击转化条件进行优化。【方法】用穿梭质粒p NW33N电转化凝结芽孢杆菌P4-102B,系统地考察高渗条件下细胞生长阶段、感受态菌体浓度、电击缓冲液组分和复苏培养基组成等因素对转化效率的影响。【结果】同一电场压力下高渗体系转化效率较低渗体系明显提高且稳定性较好,菌体在含有0.5 mol/L山梨醇的LB培养基中生长到OD600为0.8时收集,用SMG[0.5 mol/L山梨醇,0.5 mol/L甘露醇,10%(质量体积比)甘油]电击缓冲液洗涤菌体4次制备感受态,在固定电场强度14 k V/cm、脉冲时间5 ms、1 mm规格的电转杯进行电击转化,电转化后立即加入含有0.5 mol/L山梨醇和0.38 mol/L甘露醇的LB复苏培养基培养,能够获得最佳的转化效率2.7×102 CFU/μg DNA。【结论】使用高渗电击转化法能够提高电击转化的稳定性和重复性,并且可以获得较高的转化效率。     

工业谷氨酸棒杆菌电转化整合效率的优化

研究不同因素对谷氨酸棒杆菌(Corynebacterium glutamicum)电转化效率的影响,探讨电转化的最适条件,提高电转化效率。以产L-异亮氨酸的谷氨酸棒杆菌工业菌株a11为受体菌,大肠杆菌(Escherichia coli)JM109及质粒pk18mobsac B为载体,通过电转化方法研究了菌体最佳感受态性能、复苏培养基高渗溶液浓度、最适电场强度及电击后的热激培养对电转化效率的影响。对于谷氨酸棒杆菌a11而言,使用无痕的自杀载体电击转化,在感受态细胞OD_(600 nm)值为1.0~1.2,电场强度达到9.0 kV/cm,电转后46℃热激培养8 min,而且热激后继续保持37℃的适应性培养,电转化效率最高,达到1.8×10~3cfu/μg DNA。实现了工业谷氨酸棒杆菌的电转化效率的提高,也为其它谷氨酸棒杆菌电转化效率的提高提供参考方法。     

植病生防菌成团泛菌电击转化条件的研究*

研究了植病生防菌成团泛菌（Ｐａｎｔｏｅａ ａｇｇｌｏｍｅｒａｎｓ）的电击转化条件。结果表明受体菌固体培养、生长到对数早期时的细胞转化效果最好;质粒ＤＮＡ分子量增加１０培,转化效率降低１００倍;质粒ＤＮＡ浓度在０．０１－１μｇ／μＬ范围内其变化与转化频率呈正比,与转化效率成反比;从成团泛菌中提纯的质粒ＤＮＡ比从大肠杆菌中提纯的相同质粒ＤＮＡ转化成团泛菌的效率高出３０倍;最佳电击条件为场强１５ｋＶ／ｃ     

Pseudomonas stutzeri and related species undergo natural transformation

Cells of Pseudomonas stutzeri are naturally transformed by homologous chromosomal DNA; they do not require chemical treatment to become competent. This capacity to undergo natural transformation was found to be shared by the closely related species P. mendocina, P. alcaligenes, and P. pseudoalcaligenes, but was not detectable in strains of P. aeruginosa, P. perfectomarinus, P. putida, P. fluorescens, or P. syringae. P. stutzeri could be transformed either on plates or in liquid medium. Only double-stranded chromosomal DNA was effective; single-stranded DNA and plasmid DNA were not. DNA fragments larger than 10 kilobase pairs were more effective than smaller fragments. The transformation frequency was proportional to DNA concentration from 1 ng/ml to 1 microgram/ml; higher concentrations were saturating. The maximum frequency, about 10(-4) transformants per recipient cell, was obtained with cells from a culture in the early stationary growth phase. A variety of chromosomal mutations have been transformed, including mutations to auxotrophy and to antibiotic resistance. Other systems for genetic exchange in P. stutzeri have not yet been found; transformation offers a means for the genetic analysis of this metabolically versatile organism.     

Optimization of electrotransformation conditions for Propionibacterium acnes

Propionibacterium acnes has been known to be involved in the pathology of acne. However, the definite mechanism in the development of acne and the inflammation are unknown. For P. acnes, a transformation method has not been established, although it is believed to be a basic tool for gene manipulation. This study attempted to develop a P. acnes transformation method by using electroporation. Various parameters were used to develop and optimize the transformation of P. acnes. Among them two factors were crucial in the transformation for P. acnes: one was the E. coli strain from which the plasmid DNA had been isolated and the other the growth temperature of P. acnes-competent cells. It was essential to prepare plasmid DNA from a dam(-) E. coli strain, ET12567. When plasmid DNAs isolated from the other E. coli strains such as JM109 and HB101 were tested, transformation efficiency was extremely low. When P. acnes cells were cultivated at 24 degrees C for competent cell preparation, transformation efficiency increased considerably. When plasmid DNA isolated from a dam(-) mutant strain of E. coli was used for transformation of P. acnes which had been grown at 24 degrees C, maximum transformation efficiency of 1.5 x 10(4) transformants per mug of plasmid DNA was obtained at a field strength of 15 kV/cm with a pulse time of 3.2 ms. This is believed to be the first report on the transformation of P. acnes which can be employed for gene manipulations including knock-out of specific genes.     

Electrotransformation of Streptococcus agalactiae with plasmid DNA

Abstract A protocol for efficient electrotransformation of Streptococcus agalactiae (group B streptococcus) Lancefield's strain O90R (NTCT 9993) (an unencapsulated derivative of type Ia strain O90) was developed. The Escherichia coli - Streptococcus shuttle vector pDP28 (7.8 kb) carrying the ermB gene for resistance to erythromycin was used as donor DNA. Frozen 'electrocompetent' cells were prepared by repeated washes in 10% glycerol. A 50-μl aliquot containing about 5×10⁹ colony forming units of bacteria was subjected to the electric pulse. Optimal conditions for electrotransformation were determined using different media, harvesting cells at different points of the growth curve, and using different field strengths. The dose-response curve for transformation of S. agalactiae with pDP28 showed one-hit kinetics as donor DNA varied between 0.01 and 3 μg. The efficiency of electrotransformation for this range of amounts of donor DNA was 1.2×10⁴ cfu μg⁻¹. The transformation frequencies obtained with this electroporation protocol are high enough to allow both subcloning and shotgun cloning of streptococcal DNA in S. agalactiae .     

Chromosomal transformation in the cyanobacterium Agmenellum quadruplicatum.

Chromosomal transformation of Agmenellum quadruplicatum PR-6 (= Synechococcus sp. strain 7002) was characterized for phenotypic expression, for exposure time to DNA, and for dependence on DNA concentration with regard to Rifr donor DNA. Exponentially growing cells of PR-6 were competent for chromosomal transformation. Competence decreased in cells in the stationary phase of growth or in cells deprived of a nitrogen source. Dark incubation of cells before exposure to donor DNA also decreased competence. Homologous Rifr and Strr DNA and heterologous Escherichia coli W3110 DNA were used in DNA-DNA competition studies, which clearly showed that DNA binding by PR-6 was nonspecific. DNA binding and uptake by PR-6 exhibited single-hit kinetics. Single-stranded DNA failed to transform competent cells of PR-6, and DNA eclipse was not observed, suggesting that double-stranded DNA was the substrate for the binding and uptake reactions during the transformation of PR-6. A significant improvement in transformation frequency was achieved by increasing the nitrate content of the culture medium and by lowering the temperature at which cells were exposed to donor DNA from 39 degrees C (the optimal temperature for growth) to 30 degrees C.     

Natural Genetic Transformation in Monoculture Acinetobacter sp. Strain BD413 Biofilms

Horizontal gene transfer by natural genetic transformation in Acinetobacter sp. strain BD413 was investigated by using gfp carried by the autonomously replicating plasmid pGAR1 in a model monoculture biofilm. Biofilm age, DNA concentration, and biofilm mode of growth were evaluated to determine their effects on natural genetic transformation. The highest transfer frequencies were obtained in young and actively growing biofilms when high DNA concentrations were used and when the biofilm developed during continuous exposure to fresh medium without the presence of a significant amount of cells in the suspended fraction. Biofilms were highly amenable to natural transformation. They did not need to advance to an optimal growth phase which ensured the presence of optimally competent biofilm cells. An exposure time of only 15 min was adequate for transformation, and the addition of minute amounts of DNA (2.4 fg of pGAR1 per h) was enough to obtain detectable transfer frequencies. The transformability of biofilms lacking competent cells due to growth in the presence of cells in the bulk phase could be reestablished by starving the noncompetent biofilm prior to DNA exposure. Overall, the evidence suggests that biofilms offer no barrier against effective natural genetic transformation of Acinetobacter sp. strain BD413.     

DEK蛋白C末端DNA结合域的原核表达、纯化及其与DNA的结合活性

DEK蛋白C末端DNA结合域（简称CDB）是近年新发现的一个DEK蛋白与DNA的结合域,其中含有多个磷酸化位点,与DEK蛋白的功能密切相关。利用原核表达系统表达DEK蛋白的CDB肽段并进行纯化,具体为以pET30a（+）为载体质粒,E.coli BL21（DE3）为宿主细胞,构建重组基因工程菌,以IPTG诱导目的蛋白的表达,用NiNTA纯化的重组蛋白样品来进行SDSPAGE电泳分析,约在10.7kDa处出现明显的特征蛋白条带。凝胶迁移分析证实DEK蛋白C末端DNA结合域与DNA的结合倾向于与超螺旋型DNA相结合,同全长的DEK蛋白与DNA的结合具有类似的特点,表明DEK蛋白C末端DNA结合域在DEK蛋白与DNA的结合中可能具有一定的作用。     

Construction of chlorobenzene-utilizing recombinants by progenitive manifestation of a rare event.

Separate continuous cultures of Pseudomonas putida R5-3, grown on toluene, and Pseudomonas alcaligenes C-O, grown on benzoate, were concentrated and continuously amalgamated on a ceramic bead column, which was subjected to a continuous stream of chlorobenzene vapors. A recombinant strain, P. putida CB1-9, was isolated in less than 1 month. P. alcaligenes C-0 grew on benzoate and 3-chlorobenzoate but not on toluene, P. putida R5-3 grew on benzoate and toluene but not on 3-chlorobenzoate, and neither strain grew on chlorobenzene or 1,4-dichlorobenzene; however, the recombinant P. putida CB1-9 grew on all of these substrates. Chlorobenzene-utilizing strains were not found in continuous cultures run at the lowest growth rate (0.05/h) or in the absence of the donor strain, P. alcaligenes C-0. Chloride was released in stoichiometric amounts when P. putida CB1-9 was grown on either chlorobenzene or 1,4-dichlorobenzene. The recombinant strain was related to P. putida R5-3, phenotypically and genetically. Restriction enzyme digests of the single 57-kilobase (kb) plasmid in R5-3 and of the single 33-kb plasmid in CB1-9 were similar, but also indicated rearrangement of plasmid DNA. Coincidental or causal to the loss of the 24-kb fragment was the observation that the recombinant--unlike its parent, R5-3--did not grow on xylenes or methylbenzoates. Although both ortho-pyrocatechase (OP) and meta-pyrocatechase (MP) were found in CB1-9 and R5-3, MP activity was 20- to 50-fold higher in R5-3 cells grown on 4-methylbenzoate than in the same cells grown on benzene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction of chlorobenzene-utilizing recombinants by progenitive manifestation of a rare event

Separate continuous cultures of Pseudomonas putida R5-3, grown on toluene, and Pseudomonas alcaligenes C-O, grown on benzoate, were concentrated and continuously amalgamated on a ceramic bead column, which was subjected to a continuous stream of chlorobenzene vapors. A recombinant strain, P. putida CB1-9, was isolated in less than 1 month. P. alcaligenes C-0 grew on benzoate and 3-chlorobenzoate but not on toluene, P. putida R5-3 grew on benzoate and toluene but not on 3-chlorobenzoate, and neither strain grew on chlorobenzene or 1,4-dichlorobenzene; however, the recombinant P. putida CB1-9 grew on all of these substrates. Chlorobenzene-utilizing strains were not found in continuous cultures run at the lowest growth rate (0.05/h) or in the absence of the donor strain, P. alcaligenes C-0. Chloride was released in stoichiometric amounts when P. putida CB1-9 was grown on either chlorobenzene or 1,4-dichlorobenzene. The recombinant strain was related to P. putida R5-3, phenotypically and genetically. Restriction enzyme digests of the single 57-kilobase (kb) plasmid in R5-3 and of the single 33-kb plasmid in CB1-9 were similar, but also indicated rearrangement of plasmid DNA. Coincidental or causal to the loss of the 24-kb fragment was the observation that the recombinant--unlike its parent, R5-3--did not grow on xylenes or methylbenzoates. Although both ortho-pyrocatechase (OP) and meta-pyrocatechase (MP) were found in CB1-9 and R5-3, MP activity was 20- to 50-fold higher in R5-3 cells grown on 4-methylbenzoate than in the same cells grown on benzene.(ABSTRACT TRUNCATED AT 250 WORDS)