A highly efficient electroporation system for transformation of Bacillus licheniformis

Summary Transformation of intact cells of Bacillus licheniformis 5A24 with plasmids pLM6 (2.8 kb), pC194 (2.9 kb) and pCP49 (7.1 kb) by electroporation resulted in 1.5 × 10⁶, 1.2 × 10⁶ , and 5.2 × 10⁴ transformants per μg DNA, respectively. Transformation was possible with plasmid DNA, which was religated after restriction endonuclease digestions. In addition, evidence is presented that indicates that B. licheniformis possesses DNA restriction and modification systems.     

A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA

An improved method for the electrotransformation of wild-type Corynebacterium glutamicum (ATCC 13032) is described. The two crucial alterations to previously developed methods are: cultivation of cells used for electrotransformation at 18 °C instead of 30 °C, and application of a heat shock immediately following electrotransformation. Cells cultivated at sub optimal temperature have a 100-fold improved transformation efficiency (10⁸ cfu μg⁻¹) for syngeneic DNA (DNA isolated from the same species). A heat shock applied to these cells following electroporation improved the transformation efficiency for xenogeneic DNA (DNA isolated from a different species). In combination, low cultivation temperature and heat shock act synergistically and increased the transformation efficiency by four orders of magnitude to 2.5 × 10⁶ cfu μg⁻¹ xenogeneic DNA. The method was used to generate gene disruptions in C. glutamicum. Received: 26 March 1999 / Received revision: 9 June 1999 / Accepted: 11 June 1999     

简单高效的根癌农杆菌介导的水稻基因转化方法

本文在研究影响农杆菌介导的水稻转化的主要因素基础上,建立了一套简单、高效的水稻转基因系统。将水稻成熟胚来源的愈伤组织用农杆菌EHA101／pHQ9,EHA101／pHQ10,EHA101／pHQT3感染后,筛选抗性愈伤,经分化获得转化株。抗性愈伤的平均得率为约100个愈伤／g愈伤外植体,抗性愈伤的分化频率平均高达85％。转基因植株的GUS染色、Southern杂交结果表明,T-DNA上的外源基因已整合进转基因植物的基因组中。转基因植株T1代对潮霉素的抗性表明,多数转基因株系符合孟德尔分离比3：1。该系统的建立将有助于应用T—DNA标签法和基因打靶法进行水稻功能基因组的研究。     

A rapid and efficient procedure for transformation of intact Saccharomyces cerevisiae by electroporation

A rapid and efficient procedure is described for transforming Saccharomyces cerevisiae using electroporation to render intact cells permeable to DNA. The technique uses relatively low voltages and is particularly sensitive to low concentrations of plasmid DNA. At the highest voltage used (400 volts), the frequency of transformation increased with the amount of plasmid DNA between 25 ng and 100 ng. At higher concentrations of DNA (1-1.5 micrograms) electroporation yielded one-third to one-half the number of transformants obtained with a standard lithium acetate pretreatment. Because this method requires neither pretreatment of cells nor addition of polyethylene glycol (PEG), it has several advantages over currently used transformation procedures.     

一种经济高效的禾谷镰孢菌原生质体转化方法(英文)

报道一种经济高效的禾谷镰孢菌原生质体的转化方法。制备禾谷镰孢菌原生质体的最佳条件为每毫升2% Drislase和2% Snailase混合酶液中加入0.12g幼殖体,于30℃酶解1.5h。其转化效率约为40–50个转化子/μg片段DNA;转化子的PCR检测结果表明,潮霉素B基因已插入禾谷镰孢菌的基因组中。建立的禾谷镰孢菌原生质体转化系统具有经济和高效的特点,为禾谷镰孢菌基因功能的研究提供了必要的技术支持。     

A simple method for the production of highly competent cells ofAgrobacterium for transformation via electroporation

The introduction of binary plasmids intoAgrobacterium hosts forAgrobacterium-mediated transformation of plants is most readily achieved by electroporation. However, occasionally, no transformed colonies are recovered and the transformation program is delayed. Poor transformation rates are commonly associated with particular combinations ofAgrobacterium strains and plasmid-selection markers. In order to avoid this problem, it is important for the bacteria to have a highly competent status for reception of plasmid DNA. It is also important to optimize the level of antibiotic for the selection of transformed colonies. In this article, we demonstrate that transformation competence is strongly related to the phase of growth at which a bacterial culture is prepared for electroporation, and we describe a simple procedure that allows the level of transformation-competent cells to be maximized. We have observed that there is significant variation between transformedAgrobacterium strains in the levels of antibiotic tolerance; we define the antibiotic levels that are appropriate for selection of threeArgobacterium tumefaciens (EHA101, LBA4404, C58) and twoArgobacterium rhizogenes (LBA9402, Ar2626) strains, transformed with three alternative resistance markers (spectinomycin^res, kanamycin^res, and gentamycin^res).     

A rapid and highly efficient method for transformation of sugarcane callus

Modern sugarcane cultivars have complex genetic characteristics and low fertility that render their genetic improvement through traditional breeding difficult. Genetic engineering methodology to introduce foreign genes provides new opportunities for the genetic improvement of sugarcane cultivars. One of prerequisites for successful insertion of a gene cassette into the plant genome is the availability of an efficient transformation protocol. An improved protocol for Agrobacterium-mediated transformation of sugarcane is described. Between 85 and 100% of calli transformed using this procedure produced new calli, and 100% of them were positive for the inserted gene. The whole procedure permitted the production of transgenic calli in a short time (1.5 mo). The transformed calli can be cultured further for the production of the inserted gene-encoded enzyme by using cell culture, or they can be regenerated into transgenic plants. This protocol may be implemented also for the generation of transgenic plants from other species.     

A highly efficient gene-targeting system for Aspergillus parasiticus

Aims: To establish a system that greatly increases the gene‐targeting frequency in Aspergillus parasiticus. Methods and Results: The ku70 gene, a gene of the nonhomologous end‐joining (NHEJ) pathway, was replaced by the nitrate reductase gene (niaD) in A. parasiticus RHN1 that accumulates O‐methylsterigmatocystin (OMST). The NHEJ‐deficient strain, RHΔku70, produced conidia, sclerotia and OMST normally. It had identical sensitivity as RHN1 to the DNA‐topoisomerase I complex inhibitor, camptothecin, and the DNA‐damaging agent, melphalan. For targeting an aflatoxin biosynthetic pathway gene, adhA, partial restriction enzyme recognition sequences in its flanking regions were manipulated to create homologous ends for integration. Using a linearized DNA fragment that contained Aspergillus oryzae pyrithiamine resistance gene (ptr) marker the adhA‐targeting frequency in RHΔku70 reached 96%. Conclusions: The homologous recombination pathway is primarily responsible for repair of DNA damages in A. parasiticus. The NHEJ‐deficient RHΔku70, easy creation of homologous ends for integration, and the ptr‐based selection form a highly efficient gene‐targeting system. It substantially reduces the time and workload necessary to obtain knockout strains for functional studies. Significance and Impact of the Study: The developed system not only streamlines targeted gene replacement and disruption but also can be used to target specific chromosomal locations like promoters or intergenic regions. It will expedite the progresses in the functional genomic studies of A. parasiticus and Aspergilllus flavus.     

A highly efficient in vitro plant regeneration system and Agrobacterium-mediated transformation in Plumbago zeylanica

Plumbago zeylanica is a unique model for studying flowering plant gametogenesis, heterospermy, and preferential fertilization, yet understanding the control of related molecular mechanisms is impossible without efficient and reproducible regeneration and stable genetic transformation. We found three key factors for enhancing successful regeneration: (1) tissue source of explants, (2) combination and concentration of growth regulators, and (3) culture conditions. The highest frequency of shoot regeneration was achieved using hypocotyl segments cultured on MS basal medium supplemented with BA 2.0 mg/l, NAA 0.75 mg/l, adenine 50 mg/l and 10% (v/v) coconut milk under subdued light at 25±2°C; under these conditions, each hypocotyl segment produced over 30 shoots, arising primarily through direct organogenesis after 3 weeks of culture. Regenerated shoots rooted easily on half-strength basal MS medium and were successfully established in the greenhouse. Using this tissue culture protocol, reporter gene GUS under the constitutive CaMV 35S promoter was introduced into P. zeylanica cells of petiole, cotyledon and hypocotyl with A. tumefaciens strains AGL1 and LBA4404. Transient expression was observed in all recipient tissues. Stable transgenic calli originating from petiole were obtained.     

Establishment of a highly efficient transformation system for pepper (Capsicum annuum L.)

Application of modern genetic manipulation has been limited in pepper ( Capsicum annuum L.) due to the lack of an efficient transformation system. Following the development of an efficient protocol for in vitro regeneration of pepper cotyledons, we investigated the key factors affecting transformation and established a highly efficient genetic transformation system using the pepper cotyledon as starting material. In this system, cotyledon explants are preconditioned for 2 days on kanamycin (km)-free DM1 medium [Murashige and Skoog (MS) salts/Gamborg B5 vitamins basal medium supplemented with 20 g/l sucrose, 5,000 mg/l DJ nutrients and a hormone combination of 1.0 mg/l indoleacetic acid (IAA) and 5.0 mg/l 6-benzyladenine (BA) solidified with 0.7% agar, pH 5.8], followed by co-cultivation with Agrobacterium tumefaciens on DM1 for 2 days and delay selection on DM1 with 500 mg/l carbenicillin (carb) for 2 days. The explants are then placed on DM1 containing 10 mg/l AgNO(3), 50 mg/l km-sulfate and 500 mg/l carb. After 4-5 weeks, the explants with buds are transferred to EM1 medium (MS salts/Gamborg B5 vitamins basal medium supplemented with 20 g/l sucrose, 5,000 mg/l DJ nutrients, 10 mg/l AgNO(3) and a hormone combination of 1.0 mg/l IAA, 3.0 mg/l BA and 2.0 mg/l gibberellic acid, solidified with 0.7% agar, pH 5.8) with 50 mg/l kanamycin and 500 mg/l carbenicillin for the elongation of buds. After 3-6 weeks, 1- to 2-cm-long elongated shoots are excised and planted on RM1 medium (MS basal medium supplemented with a hormone combination of 0.2 mg/l NAA and 0.1 mg/l IAA, solidified with 0.8% agar, pH 5.8) with 25 mg/l km and 200 mg/l carb for rooting. We tested four genotypes of pepper, and all presented a high differentiation efficiency (81.3% on average), elongation rate (61.5%) and rooting efficiency (89.5%). Polymerase chain reaction analysis results showed that 40.8% of the regenerated plantlets were transgenic plants.     

Cell electroporation is a highly efficient method for introducing restriction endonucleases into cells

Restriction endonucleases that make either blunt- or cohesive-end DNA double-strand breaks can induce chromosome aberrations. We have used cell electroporation with great success to permeabilize Chinese hamster ovary cells for the introduction of restriction enzymes. The introduction of restriction enzymes by this method resulted in extremely high frequencies (greater than 90%) of aberrant metaphase cells and also a dramatic decrease in cell survival, as measured by subsequent colony formation. Cell electroporation by itself caused no increase in aberrant chromosomes and had only a slight effect on cell survival.     

热葡糖苷酶地芽胞杆菌NCIMB 11955高效电转感受态细胞电转体系的建立

热葡糖苷酶地芽胞杆菌(Geobacillus thermoglucosidasius)是一种革兰氏阳性兼性厌氧菌,因其高温生长速度快、不易染菌的特性,作为下一代工业生物技术的候选底盘,已被广泛用于生产生物燃料和高附加值的化学品。然而,相比于经典的模式菌株大肠杆菌(Escherichia coli),热葡糖苷酶地芽胞杆菌因其转化效率低,限制了其作为底盘菌株的高效应用。本研究的目的是开发一种能够提高该菌株电转化效率的方法。本研究通过敲除该菌株中预测的限制性内切酶基因,添加细胞壁弱化剂和细胞膜抑制剂,优化目标菌株高效感受态细胞的制备,从而提高转化效率。热葡糖苷酶地芽胞杆菌的4个限制性内切酶基因的敲除可以使感受态细胞电转化效率提高至1.2×10^(4)CFU/(μg DNA);在敲除株的基础上,分别通过添加吐温80、DL-苏氨酸和甘氨酸进一步优化感受态细胞转化效率,其中吐温80的添加使感受态细胞电转化效率提高22.5倍左右,DL-苏氨酸的添加可以使感受态细胞电转化效率增加44倍,甘氨酸的添加可以使感受态细胞电转化效率进一步提高334倍左右,优化整合添加各类试剂后感受态细胞电转化效率在限制性内切酶缺失菌株的基础上提高至4.6×10^(6)CFU/(μg DNA),相对野生型菌株提高了3个数量级。这为热葡糖苷酶地芽胞杆菌的高效遗传操作和代谢工程奠定了基础。     

Establishment of highly efficient and reproducible Agrobacterium-mediated transformation system for tomato (Solanum lycopersicum L.)

In Vitro Cellular & Developmental Biology - Plant - A simple and improved Agrobacterium-mediated transformation protocol of tomato (Solanum lycopersicum) cultivar Rio Grande was developed to...     

A highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics

We report a highly efficient protocol for the Agrobacterium-mediated genetic transformation of a miniature dwarf tomato (Lycopersicon esculentum), Micro-Tom, a model cultivar for tomato functional genomics. Cotyledon explants of tomato inoculated with Agrobacterium tumefaciens (Rhizobium radiobacter) C58C1Rif(R) harboring the binary vector pIG121Hm generated a mass of chimeric non-transgenic and transgenic adventitious buds. Repeated shoot elongation from the mass of adventitious buds on selection media resulted in the production of multiple transgenic plants that originated from independent transformation events. The transformation efficiency exceeded 40% of the explants. This protocol could become a powerful tool for functional genomics in tomato.     

Highly efficient transformation of intact yeast-like conidium cells of Tremella fuciformis by electroporation

Tremella fuciformis is one of higher basidiomycetes. Its basidiospore can reproduce yeast-like conidia, also called the blastospore by budding. The yeast-like conidia of T. fuciformis is monokaryotic and easy to culture by submerged fermentation similar to yeast. So it is a good recipient cell for exogenous gene expression. In this study, two expression vectors pGlg-gfp containing gpd-Gl promoter and gfp gene and pGlg-hph containing gpd-Gl promoter and hph gene were constructed. The lowest sensitive concentration of hygromycin for the blastospore was determined on three types of media. Our ex- periments showed that the lowest sensitive concentration of hygromycin for the blastospore was 5 μg/mL on MA medium. The intact blastospores were transformed with the expression vector pGlg-hph by electroporation. The putative transformants were obtained by the MA selective medium. Experi- mental results showed that the most effective parameters for the electroporation of intact blastospores were obtained by using STM buffer, 1.0×108 cells/mL of blastospores, 200 μL in transformation volume, 6 μg plasmid, 2.0 kV/cm of electric pulse voltage, stillness culturing on MB liquid medium for 48 h after electroporation. In these transformation conditions, the efficiency reached 277 colonies/μg DNA. Co-transformation of plasmid pGlg-gfp and pGlg-hph with ratio of 1:1 was performed by electroporation with the optimal parameters. The putative co-transformants were obtained by the MA selective medium. Eight randomly selected colonies from the vast putative co-transformants were analyzed by PCR de- tection and Southern blotting. The experiments showed that the gfp was integrated into the genomes of three transformants. The co-transformation efficiency was 37.5%. Green fluorescence was observed under laser scanning confocal microscope in these gfp positive transformants. This indicates that the exogenous gfp can be expressed effectively in the yeast-like conidia of T. fuciformis.     

A novel, highly efficient gene-cloning system for Micromonospora strains.

A highly efficient gene-cloning system for Micromonospora olivasterospora, a producer of the antibiotic fortimicin A (astromicin), suited to shotgun cloning has been developed. The system is supported by two new advancements accomplished in this study. One is the construction of novel plasmid vectors pMO116, pMO126, pMO133, pMO136, and pMO217, all consisting of replicons from newly found Micromonospora plasmids and selectable markers cloned from a neomycin-producing Micromonospora strain. The other advancement is the establishment of a new protocol for bacterial protoplasting in which some kinds of sugar alcohols are added in precultures. Such sugar alcohols were found to sensitize a wide taxonomical range of bacteria to lysozyme. The system is reproducible and reliable and has a high efficiency of more than 10(6) CFU/micrograms of DNA.     

A rapid and highly efficient sorghum transformation strategy using GRF4-GIF1/ternary vector system

Sorghum is an important crop for food, forage, wine and biofuel production. To enhance its transformation efficiency without negative developmental by-effects, we investigated the impact of GRF4-GIF1 chimaera and GRF5 on sorghum transformation. Both GRF4-GIF1 and GRF5 effectively improved the transformation efficiency of sorghum and accelerated the transformation process of sorghum to less than 2 months which was not observed when using BBM-WUS. As agrobacterium  effectors increase the ability of T-DNA transfer into plant cells, we checked whether ternary vector system can additively enhance sorghum transformation. The combination of GRF4-GIF1 with helper plasmid pVS1-VIR2 achieved the highest transformation efficiency, reaching 38.28%, which is 7.71-fold of the original method. Compared with BBM-WUS, overexpressing GRF4-GIF1 caused no noticeable growth defects in sorghum. We further developed a sorghum CRISPR/Cas9 gene-editing tool based on this GRF4-GIF1/ternary vector system, which achieved an average gene mutation efficiency of 41.36%, and null mutants were created in the T0 generation.     

A stable and efficient transformation system for Butyrivibrio fibrisolvens OB156

A 9.5-kb shuttle vector capable of replication and selection in both Escherichia coli and Butyrivibrio fibrisolvens was constructed. Plasmid pUC118 provided replication functions and ampicillin resistance selection in E. coli. In B. fibrisolvens, replication was controlled by the native plasmid pRJF1 from strain OB156, and selectability was provided by a 3.5-kb fragment of plasmid pAM1 containing the erythromycin resistance gene. Optimum conditions for transformation were 15 kV/cm, 2 h recovery, and plating in an agar overlay on medium containing 10 g erythromycin/ml. Maximum efficiency was 1.1×10⁵ transformants per g plasmid DNA (average 3×10⁴), and restriction mechanisms reduced efficiency by a factor of 2×10². Nonselective growth for 200 generations gave no measurable loss of plasmid.     

An efficient transformation system for Bifidobacterium spp.

This study describes a broad host transformation protocol that enables the uptake of plasmid DNA into 10 different species of Bifidobacterium , some of which have never been transformed before. The vector pNC7 (4·9 kb) was used to optimize the electroporation protocol. Transformation efficiencies ranged from 3·6×10⁻¹ to 1·2×10⁵ transformations per μg DNA. The impact of growth medium composition and electric field strength on transformation efficiency were independently optimized. Electrocompetent cells were grown in Iwata medium broth enriched with Actilight^RP 16%, harvested during the early exponential growth phase, and pulsed at 12·5 kV cm⁻¹, 100 Ω and 25 μF.     

A novel and highly efficient production system for recombinant adeno-associated virus vector

Recombinant adeno-associated virus(rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1(rHSV-1) designated HSV1-rc/△UL2, which expressed adeno-associated virus type2(AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein(GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/△UL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit(TU) or 4.28×104 particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy.