Development of a high-frequency transforming vector for Aspergillus nidulans

The pyr4 gene of Neurospora crassa, which codes for orotidine-5'-phosphate decarboxylase, is capable of transforming an Aspergillus nidulans pyrG mutant by chromosomal integration, despite low homology between the transforming DNA and the recipient genome. Integration of pFB6, a plasmid carrying pyr4 and capable of replication in Escherichia coli, was not observed at the pyrG locus. The efficiency of transformation was considerably enhanced (50-100 fold) by inclusion in the transforming vector of a 3.5-kb A.nidulans chromosomal sequence, ans1. Although this sequence was isolated on the basis of replicating activity in Saccharomyces cerevisiae, there was no evidence for such activity in A.nidulans. Part of the ans1 fragment appears to be reiterated in the A.nidulans genome, though it is not yet clear whether this is directly responsible for the high transformation frequency. The efficiency of transformation of A.nidulans by plasmids bearing ans1, using an improved protocol, was approx. 5 X 10(3) stable transformants per microgram of plasmid DNA.     

Sequence and centromere proximal location of a transformation enhancing fragment ans1 from Aspergillus nidulans.

The Aspergillus nidulans sequence ans1, previously known to enhance transformation frequencies of pyr4-based vectors, was shown to enhance the efficiency of argB and trpC-based vectors. Increased efficiencies could be obtained by constructing vectors containing argB and ans1 or by cotransforming selectable plasmids (containing argB, trpC, or pyr4) with the non-selectable ans1 sequence. The preponderance of evidence suggests that the mechanism of ans1 activity does not involve homologous recombination events, in spite of the presence of multiple regions of homology in the A. nidulans genome. Genetic mapping localized ans1 to the vicinity of the centromere of linkage group I. The nucleotide sequence of a 1.8 Kb functional subclone of ans1 was determined and found to be highly A + T rich (81%).     

Restriction-like phenomena in transformation of Bacillus subtilis recA.

Genetic transformation in recA1 strains of Bacillus subtilis was studied to test the hypothesis that, in these strains, a major pathway of recombination is missing, leaving only residual transformation via a pathway specific for transduction. The two putative recombinational pathways have been hypothesized to differ in either length of synapsed regions or specificity for nucleotide sequence homology. It was found that the efficiency of transformation of recA1 cells by deoxyribonucleic acid (DNA) from the heterologous strain W23 was much lower than when a homologous donor DNA was used, the relative efficiency being different for different genetic markers. Because the frequency of recombination between linked markers is only slightly changed in recA1 recipients, and because markers of heterologous origin in DNA from intergenotic strains are not discriminated against strongly by recA1 recipients, it is concluded that neither a difference in length of synapsed DNA nor a difference in specificity for nucleotide sequence homology accounts for reduced transformation in recA1 cells. It is proposed that at some time between uptake and integration, heterologous DNA is inactivated by restriction, and that aberrant restriction of repaired regions may account for reduced transformation by homologous DNA.     

High efficiency transformation by electroporation of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Yarrowia lipolytica was usually transformed by heat shock, but linearized integrative vectors always resulted in a low transformation efficiency when electroporation was used. To develop a high efficiency integrative transformation method by electroporation of F. lipolytica, we report here that pretreatment of F. lipolytica with 150 mM LiAc for 1 h before electroporation will approximately 30-fold of increase transformation efficiency. A cell concentration of 10¹⁰/ml and instrument settings of 1.5 kV will generate the highest transformation efficiencies. We have developed a procedure to transform F. lipolytica that will be able to yield an efficiency of 2.1 × 10⁴ transformants/ug for integrative linear DNA. With our modifications, the electroporation procedures became a very efficient and reliable tool for F. lipolytica transformation.     

Bacterial transformation using micro-shock waves

Shock waves are one of the most competent mechanisms of energy dissipation observed in nature. We have developed a novel device to generate controlled micro-shock waves using an explosive-coated polymer tube. In this study, we harnessed these controlled micro-shock waves to develop a unique bacterial transformation method. The conditions were optimized for the maximum transformation efficiency in Escherichia coli. The maximum transformation efficiency was obtained when we used a 30 cm length polymer tube, 100 μm thick metal foil, 200 mM CaCl₂, 1 ng/μl plasmid DNA concentration, and 1 × 10⁹ cell density. The highest transformation efficiency achieved (1 × 10⁻⁵ transformants/cell) was at least 10 times greater than the previously reported ultrasound-mediated transformation (1 × 10⁻⁶ transformants/cell). This method was also successfully employed for the efficient and reproducible transformation of Pseudomonas aeruginosa and Salmonellatyphimurium. This novel method of transformation was shown to be as efficient as electroporation with the added advantage of better recovery of cells, reduced cost (40 times cheaper than a commercial electroporator), and growth phase independent transformation.     

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.     

Genetic transformation of Chainia and heat attenuation of its restriction system.

A PEG-mediated transformation system for Chainia (NCL 82-5-1) was developed using a broad host range Streptomyces vector, pIJ702. Protoplasts prepared from Chainia (NCL 82-5-1) were regenerated with 5% efficiency. Transformation of the protoplasts with pIJ702 gave 10-20 transformants/micrograms DNA. The low efficiency of transformation is attributed to a restriction system in Chainia; this could be inhibited by treating the protoplasts at 42 degrees C for 10 min just before transformation. The yield of transformants increased 100-fold when pIJ702 was modified by passage in Chainia. Because the plasmid replicon was functional in Chainia and the modified plasmid was stably maintained, the transformation system should be useful for self-cloning in Chainia NCL 82-5-1 of the many commercially important enzymes this strain is known to produce.     

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.     

Microwave improved Escherichia coli transformation

Aims: The calcium chloride chemical transformation of Escherichia coli is still the most widley used cloning method in small laboratories. Therefore, any practicable improvement in its transformation efficiency seems to be of general interest. Methods and Results: We found that giving calcium chloride competent cells a 1 min microwave pulse at the lowest power setting (180 W), instead of the classic 1–2 min 42°C heat‐shock step, increases the transformation efficiency around threefold (3.3 ± 0.5). Moreover, when both treatments were given in a 2‐min 42°C ? 5 min on ice ? 1 min microwave pluse sequence, an additional improvement of 1.6 was obtained, resulting in an overall increase in efficiency of approximately 5.3‐fold compared to classical heat shock. Conclusions: This transformation method significantly improves the classical heat shock treatment. Significance and Impact of the Study: This method might be useful to those laboratories that cannot afford an electroporation apparatus.     

Protamine-mediated DNA coating remarkably improves bombardment transformation efficiency in plant cells

We have developed a method by which remarkably higher efficiencies of transient and stable transformation were achieved in bombardment transformation of plants. Over fivefold increase in transient gus gene expression was achieved when rice or maize suspension cells were bombarded with gold particles coated with plasmid DNA in the presence of protamine instead of the conventional spermidine. A 3.3-fold improvement in stable transformation efficiency was also observed using rice suspension cells with the new coating approach. The coated protamine-plasmid DNA complex resisted degradation by a DNase or by rice cell extract much longer than the spermidine-plasmid DNA complex. The results from this study suggest that protamine protects plasmid DNA longer than spermidine when being delivered inside the cells, probably by forming a nano-scale complex, and thus helps improve the efficiency of particle bombardment-mediated plant transformation.     

Cell-cycle dependent transformation competence in Dictyostelium discoideum

We describe a modification of the transformation procedure for Dictyostelium which allows for a more exact estimate of transformation efficiency and the isolation of primary transformants. Investigations of transformation competence revealed a negative correlation to cell density and a distinct distribution during the cell-cycle. In synchronized cells, transformation efficiency is 2-3 fold higher during mitosis when compared to unsynchronized cells.     

Restriction of DNA encoding selectable markers decreases the transformation efficiency of Helicobacter pylori

Helicobacter pylori populations recovered from the human stomach display extensive recombination and quasispecies development, and this suggests frequent exchange of DNA between different strains in vivo. In vitro, however, most H. pylori strains display restriction to the uptake of non-self DNA, as measured using selectable markers, regardless of their competency for transformation with self DNA. We have examined the effect of different selectable markers on double-crossover recombination efficiencies in three reference strains (1061, 26695 & SS1) and one clinical isolate (CHP1) of H. pylori. All strains were efficiently transformable to kanamycin or chloramphenicol resistance by using self-genomic DNA from isogenic mutants bearing the aphA3 or cat cassettes, respectively. However, strains 26695 and CHP1 showed a 3-5-log reduction in transformation efficiency by non-self recombinant DNA containing aphA3, when compared to cat. Strain 1061 readily accepted either cassette, and strain SS1 was poorly tolerant of any non-self DNA. Genome-wide random mutagenesis of these strains was only achievable with a selectable marker that allowed high transformation efficiency. Digestion of 32P-labelled cassettes by H. pylori lysates mirrored the transformation results and indicated that in some strains these cassettes are the targets of enzymatic restriction.     

Expression of the trpC gene from Penicillium chrysogenum in Aspergillus nidulans

The heterologous expression in Aspergillus nidulans of a gene involved in tryptophan biosynthesis from Penicillium chrysogenum is described. With the chimeric plasmid pPC-31, which carries the cloned trpC gene, approximately 10-40 "stable" transformants per microgram of DNA were obtained, with selection for complementation of the mutant allele. This frequency was increased 10-fold by the insertion of the ans1 fragment into the transformation vector. Southern hybridization analysis revealed that transformation occurred as a consequence of the integration of vector sequences into the host chromosome at a variety of sites within the genome.     

Nucleosomes affect local transformation efficiency

Genetic transformation is a natural process during which foreign DNA enters a cell and integrates into the genome. Apart from its relevance for horizontal gene transfer in nature, transformation is also the cornerstone of today''s recombinant gene technology. Despite its importance, relatively little is known about the factors that determine transformation efficiency. We hypothesize that differences in DNA accessibility associated with nucleosome positioning may affect local transformation efficiency. We investigated the landscape of transformation efficiency at various positions in the Saccharomyces cerevisiae genome and correlated these measurements with nucleosome positioning. We find that transformation efficiency shows a highly significant inverse correlation with relative nucleosome density. This correlation was lost when the nucleosome pattern, but not the underlying sequence was changed. Together, our results demonstrate a novel role for nucleosomes and also allow researchers to predict transformation efficiency of a target region and select spots in the genome that are likely to yield higher transformation efficiency.     

A high-throughput <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation system for the grass model species <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> L.

In the ongoing process of developing Brachypodium distachyon as a model plant for temperate cereals and forage grasses, we have developed a high-throughput Agrobacterium-mediated transformation system for a diploid accession. Embryogenic callus, derived from immature embryos of the accession BDR018, were transformed with Agrobacterium tumefaciens strain AGL1 carrying two T-DNA plasmids, pDM805 and pWBV-Ds-Ubi-bar-Ds. Transient and stable transformation efficiencies were optimised by varying the pre-cultivation period, which had a strong effect on stable transformation efficiency. On average 55% of 17-day-old calli co-inoculated with Agrobacterium regenerated stable transgenic plants. Stable transformation frequencies of up to 80%, which to our knowledge is the highest transformation efficiency reported in graminaceous species, were observed. In a study of 177 transgenic lines transformed with pDM805, all of the regenerated transgenic lines were resistant to BASTA((R)), while the gusA gene was expressed in 88% of the transgenic lines. Southern blot analysis revealed that 35% of the tested plants had a single T-DNA integration. Segregation analysis performed on progenies of ten selected T(0) plants indicated simple Mendelian inheritance of the two transgenes. Furthermore, the presence of two selection marker genes, bar and hpt, on the T-DNA of pWBV-Ds-Ubi-bar-Ds allowed us to characterize the developed transformation protocol with respect to full-length integration rate. Even when not selected for, full-length integration occurred in 97% of the transformants when using bialaphos as selection agent.     

少量制备大肠杆菌感受态细胞条件探索

目的:为了获得重复性好、转化率高的少量制备感受态细胞的方法,利用不同生长时期的大肠杆菌感受态细胞,进行转化比较。方法:根据普通实验室的实验条件,常规方法提取质粒,氯化钙法转化不同生长时期的大肠杆菌感受态细胞,比较转化率。结果:大肠杆菌感受态细胞的转化率与OD值显著相关,在OD600nm为0.39和0.55时转化率最高,在OD600nm为0.28~0.55之间均可得到理想的转化效果。结论:少量制备感受态细胞方法操作中无需添加任何保护试剂和细胞复苏培养,操作简便、重复性好,实验成本低廉。     

High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol

Transformation efficiencies for Pichia pastoris are usually several orders of magnitude below those for other yeast. We report here that pretreatment of P. pastoris with 0.1 M lithium acetate (LiAc) and 10 mM dithiothreitol (DTT) before electroporation increased transformation efficiency approximately 150-fold. DTT alone enhanced the transformation efficiency up to 20-fold, but LiAc alone had little effect. Cultures grown to 1.15-2.6 A at 600 nm had higher transformation efficiencies than younger or older cultures. A cell concentration of 10(10)/mL gave the highest efficiencies. Digestion of pPIC9K within the AOX1 gene with Sacl gave efficiencies approximately 30 times higher than digestion in other genes with other enzymes. Given the optimization of these factors, the highest transformation efficiency was obtained with instrument settings of 1.5 kV, 25 microF, and 186 omega. The transformation efficiency at optimal conditions reached 4 x 10(6) transformants/microgram DNA with pPIC9K. A maximum of 2.6 x 10(5) transformants was produced when 1 microgram of pPIC9K DNA was used.     

Tn<Emphasis Type="Italic">5</Emphasis> transposase-assisted high-efficiency transformation of filamentous fungus <Emphasis Type="Italic">Phoma herbarum</Emphasis> YS4108

Conventionally, filamentous fungi are transformed by using conidia or protoplasts as recipients. However, induction of sporulation is difficult in some fungi, and protoplasting is an awing, frequently frustrating, and batch-dependent work. In this study, we established a simple and convenient method to prepare single cells from mycelia without enzymatic protoplasting. As a case study on the pathogenic fungus Phoma herbarum YS4108, the single cells could be directly and highly efficiently transformed with the aid of Tn5 transposase. The optimal electric pulse delivery parameters were 25 muF in capacitance, 0.75 kV (0.2-cm cuvette) in voltage, and 400 Omega in resistance, under which the efficiency of transposase-assisted transformation (TNAT) was enhanced to two to threefold compared to that of non-TNAT method, resulting in >230 transformants/cuvette (10(6) recipients). Further cell wall weakening of the single cells by lytic enzymes and linearization of the plasmid were found to have no effects on transformation efficiency, but vector linearization apparently lowered the background growth. The present study for the first time explained that Tn5 transposase could be used to increase transformation efficiency in filamentous fungi, and the method presented here may be of wide applicability in different studies and may be the first choice when transformation efficiency and convenience are priorities and mycelia have to be used as transformation recipients.     

Improved method for electroporation of Staphylococcus aureus

We have developed a significantly improved method for the electroporation of plasmid DNA into Staphylococcus aureus. The highest transformation efficiency achieved with this procedure was 4.0 x 10(8) transformants per microgram of plasmid pSK265 DNA. This represents a 530-fold improvement over the previously reported optimum efficiency of 7.5 x 10(5) transformants per microgram of plasmid DNA after electroporation of S. aureus cells [9]. Identical results were obtained when electrocompetent cells, which had been stored frozen at -80 degrees C, were used. The improved efficiency is due primarily to the use of a modified medium (designated as B2 medium) and secondarily to the use of 0.1-cm cuvettes. Several other plasmids (pI258, pMH109, and pSK270) were also electrotransformed into competent cells using our procedure, and for each plasmid, the transformation efficiency was significantly reduced compared to that observed when pSK265 DNA was used. With respect to plasmid pI258, the transformation efficiency was 3500-fold higher than that reported previously for transformation of this plasmid into S. aureus RN4220 [9]. The optimized electroporation procedure was less successful in transforming other staphylococci. Electrocompetent cells of S. aureus ATCC 29213 and S. epidermidis ATCC 12228 produced 5.5 x 10(5) and 5 x 10(3) transformants per microgram of pSK265 DNA, respectively.