Role of membrane potential on artificial transformation of E. coli with plasmid DNA

The standard method of transformation of Escherichea coli with plasmid DNA involves two important steps: cells are first suspended in 100mM CaCl(2) at 0 degrees C (in which DNA is added), followed by the administration of a heat-pulse from 0 to 42 degrees C for 90s [Cohen, S., Chang, A., Hsu, L., 1972. Nonchromosomal antibiotic resistance in bacteria. Proc. Natl. Acad. Sci. U.S.A., 69, 2110-2114]. The first step makes the cells competent for uptake of DNA and the second step is believed to facilitate the DNA entry into the cells by an unknown mechanism. In this study, the measure of membrane potential of the intact competent cells, at different steps of transformation process, either by the method of spectrofluorimetry or that of flow cytometry, indicates that the heat-pulse step (0-->42 degrees C) heavily decreases the membrane potential. A subsequent cold shock (42-->0 degrees C) raises the potential further to its original value. Moreover, the efficiency of transformation of E. coli XL1 Blue cells with plasmid pUC19 DNA remains unaltered when the heat-pulse step is replaced by the incubation of the DNA-adsorbed competent cells with 10 microM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 90s at 0 degrees C. Since the CCCP, a well-known protonophore, reduces membrane potential by dissipating the proton-motive-force (PMF) across E. coli plasma membrane, our experimental results suggest that the heat-pulse step of the standard transformation procedure facilitates DNA entry into the cells by lowering the membrane potential.     

Recombination following transformation of Escherichia coli by heteroduplex plasmid DNA molecules

Circular heteroduplex DNA molecules introduced into Escherichia coli-competent cells are converted to new recombinant plasmids as a result of enzymatic actions in vivo. A pair of plasmids with partial sequence homology were each linearized at a different position with restriction enzymes, and the termini were made flush with the single-strand-specific S1 nuclease. Duplex molecules were then formed by melting and annealing these plasmid DNAs together. In contrast to linear homoduplex molecules, heteroduplexes circularize and therefore transform E. coli efficiently. Unique DNA sequences on each of the parental strands in the transforming heteroduplexes can be selectively incorporated or deleted as a result of in vivo enzymatic activities in transformed cells. This method permits the generation of new recombinant sequences in vivo without relying solely on the presence of convenient restriction sites for manipulation of DNA fragments in vitro.     

Simultaneous transformation of Escherichia coli by pairs of compatible and incompatible plasmid DNA molecules.

Summary This paper describes experiments involving simultaneous transformation of Escherichia coli by DNA of two species of multicopy plasmid in order to study competence and DNA uptake in this organism. In transformation mixtures where separate species of plasmid DNA were present in equal amounts, selection for a single plasmid gave doseresponse curves with slopes of 1. These results indicate that uptake of a single molecule of plasmid DNA is sufficient to produce one transformant. Simultaneous selection for markers on both plamsids gave a dose-response curve with a slope of 2. The total numbers of transformants obtained in single or double transformation experiments at saturating DNA concentrations were the same, and represented the maximum number of transformable cells. However, the absolute frequency of double transformants was reduced 4–6 fold relative to frequencies obtained with single markers. Similar results were obtained using pairs of compatible or incompatible plasmids in rec ⁺ or recA strains. These findings may be explained either on the basis of interference between competing plasmid molecules for uptake or establishment or by assuming that competent cells of Escherichia coli vary in their ability to incorporate more than one plasmid DNA molecule. Our results are consistent with an interpretation where more than 80% of the transformable cells are only capable of establishing one plasmid moiecule.     

Chrysotile asbestos fibers mediate transformation of Escherichia coli by exogenous plasmid DNA

The ability of chrysotile asbestos fibers to introduce the exogenous plasmid pUC18 into Escherichia coli JM109 cells was tested. Cells were transformed with pUC18 DNA although the frequency of transformation was quite low: 759+/-301 transformants were obtained per microgram of pUC18. Plasmids were purified from E. coli which had been transformed by mediation with chrysotile asbestos. Following this, the plasmids were confirmed to be pUC18 by Southern hybridization. This asbestos-mediated transformation was optimal within 5 min when 10 mg ml(-1) of asbestos was used. Plasmids up to 7.69 kb were introduced by this method.     

Intramolecular recombination during plasmid transformation of Bacillus subtilis competent cells.

We have constructed plasmids carrying direct internal repeats 260-2000 bp long. Monomers of such plasmids transformed Bacillus subtilis competent cells. The efficiency of transformation varied with the square of the length of repeats. The transformed clones harbored either the entire transforming plasmid and the plasmid arising by recombination between the repeats, or only the latter plasmid. Internally-repeated plasmids linearized by in vitro cleavage with restriction endonuclease could transform, yielding clones which exclusively harbored a plasmid resulting from recombination between the repeats. When the transforming plasmid carried repeats which differed slightly, conversion of one repeat into the other could occur. The following model of plasmid transformation accounts for these data: (1) plasmid DNA is cleaved and rendered linear in contact with competent cells; (2) a linear, at least partially double-stranded plasmid molecule is introduced or formed by repair within the cell; (3) a circular viable plasmid is produced by recombination between repeats carried on this molecule; (4) alternatively, a viable plasmid is produced by repairing the cut within one of the repeats by DNA synthesis which uses the other repeat as a template.     

Deletion and rearrangement of plasmid DNA during transformation of Escherichia coli with linear plasmid molecules.

When E. coli was transformed with linearized pBR322 DNA, many transformants contained recircularized plasmids bearing deletions and other rearrangements. Most aberrant molecules were less than monomeric length and had lost the restriction site used for linearization, with the deleted region extending mono- (type Ia) or bi-directionally (type Ib). Type II deletants were greater than monomeric but less than dimeric and contained the pBR322 sequence in direct repeat with deletion at one or both junctions (type IIa) or in inverted repeat with loss of sequence at both junctions (type IIb). Type III deletants were greater than dimeric but less than trimeric, consisting of pBR322 sequences in both direct and inverse repeat with deletions at two or more junctions. Transformation frequencies for linear DNA were drastically reduced in xth-1- bacteria with type IIb deletants predominating in transformants. This indicates that exonuclease III is important for perfect recyclization of plasmids and the generation of type I deletants. In vivo recyclization of in vitro ligation products explains many of the aberrant DNA molecules that are encountered during gene cloning.     

Genetic transformation of Rhizobium leguminosarum by plasmid DNA.

We demonstrated the genetic transformation of Rhizobium leguminosarum by R68.45 plasmid DNA by freezing and thawing cell suspensions in the presence of R68.45 plasmid DNA and 20 mM MgCl2. Clones resistant to kanamycin and tetracycline were recovered at a frequency of 10(-8) per recipient cell. No colonies that were doubly drug resistant were recovered in parallel control experiments.     

High-efficiency transformation of mammalian cells by plasmid DNA.

We describe a simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells. In this protocol, the calcium phosphate-DNA complex is formed gradually in the medium during incubation with cells and precipitates on the cells. The crucial factors for obtaining efficient transformation are the pH (6.95) of the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation of the DNA with the cells, and the amount (20 to 30 micrograms) and the form (circular) of DNA. In sharp contrast to the results with circular DNA, linear DNA is almost inactive. Under these conditions, 50% of mouse L(A9) cells can be stably transformed with pcDneo, a simian virus 40-based neo (neomycin resistance) marker vector. The NIH3T3, C127, CV1, BHK, CHO, and HeLa cell lines were transformed at efficiencies of 10 to 50% with this vector and the neo marker-incorporated pcD vectors that were used for the construction and transduction of cDNA expression libraries as well as for the expression of cloned cDNA in mammalian cells.     

Effect of the molecular weight of DNA on the effectiveness of plasmid transformation of E. coli K12

The level of plasmid transformation and transfection by the high molecular mass DNA was studied for Escherichia coli mutants having increased efficiency of plasmid transformation by low molecular mass DNA. Decreased level of plasmid transformation and transfection registered in some mutants as compared to the one in wild type strain suggests the specificity of Escherichia coli cells penetration for DNA of different molecular mass.     

Genetic transformation of Rhodopseudomonas sphaeroides by plasmid DNA.

A broad-host-range cloning vector, pUI81, was constructed in vitro from plasmids RSF1010 and pSL25 (a pBR322 derivative) and used to assay for transformation in Rhodopseudomonas sphaeroides. Washing cells with 500 mM Tris was an effective means of inducing competence for DNA uptake. Transformation frequencies as high as 10(-5) (transformants per viable cell) have been achieved by incubating Tris-treated cells with plasmid DNA, 100 mM CaCl2, and 20% polyethylene glycol 6000. Maximum frequencies were obtained when recipient cells were spread onto selective media after a 6.5-h outgrowth period in antibiotic-free medium. The structure (open circular versus closed, covalent circular), size, and concentration of plasmid DNA all significantly affected the transformation frequency. Four different plasmids, all small and suitable as cloning vectors, have been introduced by transformation into several different R. sphaeroides strains. Recombinant DNA carried on small, nonconjugative plasmids with broad host ranges can now be directly transferred to R. sphaeroides by this method.     

Optimization of routine transformation of Escherichia coli with plasmid DNA

Methods to optimize resources and transformation efficiency of routine daily transformations of DH1 Escherichia coli prepared by three calcium chloride methods were investigated and compared with polyethylene glycol and Hanahan methods. The benefit of a heat-shock step, a preplating incubation step to allow expression of antibiotic resistance, use of log phase bacteria and prolonged storage of bacteria were investigated using pBR322 and pUC18 plasmid DNAs. Bacteria prepared by CaCl2 methods consistently gave efficiencies of 4 x 10(6) transformants/microgram of plasmid DNA or better and were overall the most labor- and resource-efficient methods. Use of log phase bacteria, a heat shock and an incubation step were found to be beneficial for freshly prepared bacteria for all methods. Prolonged storage of up to 30 days of bacteria prepared by the CaCl2 methods was beneficial, resulting in a sustained increase in transformation efficiency when selection was by ampicillin but not when by tetracycline resistance. Also found when using bacteria stored three days or longer was an increased transformation efficiency of stationary vs. log phase bacteria and an unchanged or even increased efficiency when the preplating incubation step was omitted. The Hanahan methods were the most labor and resource intensive and routinely gave efficiencies of 2 x 10(7). Higher efficiencies of 10(8) were obtained only with repeated trial and error and were not consistently reproducible. The polyethylene glycol method consistently gave efficiencies of 2 x 10(7), and bacteria could easily be prepared daily or frozen with a minimal decrease in efficiency.     

保存时间及温度对大肠杆菌感受态转化效率的影响

通过不同保存时间和温度对大肠杆菌感受态转化效率影响的研究,更明确阐明了不同保存温度下感受态细胞随保存时间的延长其转化率的变化趋势,为以后的研究提供了量化的指标。     

大肠杆菌质粒DNA PCR模板的快速制备

在分子遗传学的研究中要频繁地用到ＰＣＲ的方法。本文基于粗糙脉孢霉的方法[1] 发展了快速制备大肠杆菌质粒ＤＮＡＰＣＲ模板的程序 ,具有良好的重复性 ,为分子遗传学研究提供了快速有效的方法。收稿日期 :2 0 0 1 - 1 0 - 2 0 ;修回日期 :2 0 0 1 - 1 1 - 2 1基金项目 :广东省自然科学基金 (Ｎｏ.980 30 3) ;国家自然科学基金 (Ｎｏ .30 0 70 4 2 0 ) ;教育部高等学校骨干教师计划资助项目 (Ｎｏ.2 0 0 0 - 0 61 -31 4 30 1 )作者简介 :罗樨 (1 977- ) ,女 ,硕士 ,从事生物钟研究。图 1　质粒ＤＮＡＰＣＲ扩增结果1 ＤＬ2 0 0 0分子量标…     

Intergeneric natural plasmid transformation between E. coli and a marine Vibrio species

Natural transformation is the mechanism of procaryotic gene transfer that involves the uptake and expression of genetic information encoded in extracellular DNA. This process has been regarded as a mechanism to transfer genes (primarily chromosomal markers) between closely related strains or species. Here we demonstrate the cell-contact-dependent transfer of a non-conjugative plasmid from a laboratory E. coli strain to a marine Vibrio species, the first report of intergeneric natural plasmid transformation involving a marine bacterium. The nucleic acid synthesis inhibitors nalidixic acid and rifampicin inhibited the ability of the E. coli to function as a donor. However, dead cells also served as efficient donors. There was an obligate requirement for cell contact. No transfer occurred in the presence of DNase I, when donors and recipients were separated by a 0.2-micron filter, or when spent medium alone was used as a source of transforming DNA. These results indicate that contact-mediated intergeneric plasmid exchange can occur in the absence of detectable viable donor cells and that small non-conjugative plasmids can be spread through heterogeneous microbial communities by a process previously not recognized, natural plasmid transformation. These findings are important in the assessment of genetic risk to the environment, particularly from wastewater treatment systems and the use of genetically engineered organisms in the environment.