Microprojectile bombardment as a reliable method for transformation of the mucoralean fungus Absidia glauca

Genetic analysis of all Mucor-like fungi is severely impaired by the low efficiency of transformation systems and the genetic instability of the introduced plasmid constructs. The transformation efficiency of one of the model systems among mucoralean fungi, Absidia glauca, was improved considerably by microprojectile bombardment. For this purpose, a plasmid was constructed conferring (i) neomycin resistance as a selective marker and (ii) fluorescence due to expression of the gfp gene from the jellyfish Aequorea victoria. Compared with previous techniques, this method offers increased efficiency, with considerably easier handling than procedures based on protoplasts and, therefore, improved reliability. The uninucleate sporangiospores of A. glauca can be transformed early during the germination process. At this stage the number of nuclei ranges between 1 and 2. Thus, the abundance of transgenic nuclei in the coenocytic mycelia is high, and fewer problems are encountered with detecting low expression levels of the genes used for selection and monitoring of transformants.     

Transient expression of the GUS gene in a unicellular marine green alga,<Emphasis Type="Italic">Chlorella</Emphasis> sp.<Emphasis Type="Italic">MACC/C95</Emphasis>, via electroporation

A transient expression system for a unicellular marine green alga,Chlorella sp.MACC/C95, was developed using a reporter GUS gene coded for by plasmid pBI121. The results demonstrated a high transformation efficiency could be achieved by using electroporation to deliver DNA into intact cells and the CaMV35S promoter to drive the foreign gene expression inChlorella sp.MACC/C95. The use of a carrier DNA coupled with osmosis treatment improved the transformation efficiency, while linearization of the plasmid had minor effects. Investigation of the effects of DNA concentration and growth phases ofChlorella sp.MACC/C95 on transformation efficiency indicated that the highest level of transient expression was observed when 6 μg mL⁻¹ of plasmid DNA and cells 2–6 days old were used.     

Plasmid‐free T7‐based Escherichia coli expression systems

In order to release host cells from plasmid‐mediated increases in metabolic load and high gene dosages, we developed a plasmid‐free, T7‐based E. coli expression system in which the target gene is site‐specifically integrated into the genome of the host. With this system, plasmid‐loss, a source of instability for conventional expression systems, was eliminated. At the same time, system leakiness, a challenging problem with recombinant systems, was minimized. The efficiency of the T7 RNA polymerase compensates for low gene dosage and provides high rates of recombinant gene expression without fatal consequences to host metabolism. Relative to conventional pET systems, this system permits improved process stability and increases the host cell's capacity for recombinant gene expression, resulting in higher product yields. The stability of the plasmid‐free system was proven in chemostat cultivation for 40 generations in a non‐induced and for 10 generations in a fully induced state. For this reason plasmid‐free systems benefit the development of continuous production processes with E. coli. However, time and effort of the more complex cloning procedure have to be considered in relation to the advantages of plasmid‐free systems in upstream‐processing. Biotechnol. Bioeng. 2010. 105: 786–794. © 2009 Wiley Periodicals, Inc.     

Transformation in Heterokaryons of <Emphasis Type="Italic">Neurospora crassa</Emphasis> Is Nuclear Rather Than Cellular Phenomenon

In Neurospora crassa, multinucleate macroconidia are used for genetic transformation. The barrier for such a transformation can be either at the cell membrane level or at the nuclear membrane level. For assessment of these possibilities, a forced heterokaryon (containing two genetically marked nuclei and auxotrophic for histidine) of Neurospora crassa was transformed with a plasmid containing his-3 ⁺ gene. The transformants, which could grow without histidine supplementation, were then resolved into component homokaryons to determine into which nucleus or nuclei the plasmid had entered. Our results suggest that the barrier for transformation in Neurospora crassa is at the nuclear level, not at the cell membrane level. In a heterokaryon containing two genetically distinct nuclei, plasmid DNA integrated into only one of the nuclear types at any instance, but never into both nuclear types. Thus, in Neurospora crassa, the competent nucleus is essential for the transformation event to take place, and at a given time only one type of nucleus is competent to take up the exogenous DNA. Genomic Southern analysis showed that the transformants harbor both ectopic and homologous integrations of the plasmid DNA. The type and number of integrations were reflected at the post-translational level, since the specific activity of histidinol dehydrogenase (the translation product of his-3 ⁺ gene) was variable among several transformants and always less than the level of the wild type. Received: 24 July 2001 / Accepted: 15 August 2001     

Factors affecting transformation efficiency of embryogenic callus of Upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) with <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

A reliable and high-efficiency system of transforming embryogenic callus (EC) mediated by Agrobacterium tumefaciens was developed in cotton. Various aspects of transformation were examined in efforts to improve the efficiency of producing transformants. LBA4404 and C58C3, harboring the pgusBin19 plasmid containing neomycin phosphortransferase II (npt-II) gene as a selection marker, were used for transformation. The effects of Agrobacterium strains, acetosyringone (AS), co-cultivation temperature, co-cultivation duration, Agrobacterium concentration and physiological status of EC on transformation efficiency were evaluated. Strain LBA4404 proved significantly better than C58C3. Agrobacterium at a concentration of 0.5 × 10⁸ cells ml^–1 (OD₆₀₀=0.5) improved the efficiency of transformation. Relatively low co-cultivation temperature (19 °C) and short co-cultivation duration (48 h) were optimal for developing a highly efficient method of transforming EC. Concentration of AS at 50 mg l^–1 during co-cultivation significantly increased transformation efficiency. EC growing 15 days after subculture was the best physiological status for transformation. An overall scheme for producing transgenic cotton is presented, through which an average transformation rate of 15% was obtained.     

Extranuclear Expression of the Bacterial Xylose Isomerase (xylA) and the UDP-Glucose Dehydrogenase (hasB) Genes in Yeast with Kluyveromyces lactis Linear Killer Plasmids as Vectors

On the basis of the linear killer plasmid pGKL1 from Kluyveromyces lactis, two new linear hybrid plasmids were constructed. One of these, pRSC126, carried the xylA gene from Streptomyces rubiginosus encoding the xylose isomerase. The other linear hybrid molecule, pRSC128, carried the hasB gene of Streptococcus pyogenes encoding the UDP glucose dehydrogenase. Construction was performed in a way that the putative cytoplasmic promoter element of ORF5 of pGKL2 was fused to the coding region of the heterologous genes. After transformation, in vivo recombination led to the establishment of linear hybrid vectors. Though efficiency of expression was low when compared with bacterial systems, cytoplasmic expression of both genes was clearly demonstrated. Received: 1 April 1996 / Accepted: 30 May 1996     

Structural Elucidation of Alterporriol B,a Novel Metabolic Pigment Produced by Alternaria porri (Ellis) Ciferri

Restriction-reduced mutants were isolated from Streptomyces rosa subsp. notoensis KA301 and S. tanashiensis strain Kala which produce the benzoisochromanequinone antibiotics nanaomycin and kalafungin, respectively. The mutants of S. rosa, which can be transformed with a multi-copy plasmid and in which the actinophage Pa16 can propagate, were selected. They were transformed with a single-copy plasmid propagated in S. lividans TK24, and with its modified plasmid propagated in the mutant at higher efficiency. The mutants of S. tanashiensis were selected by their capability to be transformed with a multi-copy plasmid. The efficiency of transformation with a single-copy plasmid propagated in S. lividans TK24 was low, but was much increased by heating the protoplasts at 42°C for 15 min prior to the transformation. These mutants derived from both strains probably lack at least one of their restriction systems.     

Staurosporine,a Prolyl Endopeptidase Inhibitor

The filamentous fungus Monascus pilosus was genetically transformed with a reporter plasmid, pMS-1.5hp, by aurintricarboxylic acid (ATA) treatment to obtain an efficient red-pigment producing mutant. The transformation efficiency of Monascus pilosus was higher with the ATA-treatment than with either a non-restriction-enzyme-mediated integration (REMI) or a REMI method. This valid and convenient random mutagenesis method shows that ATA can be applied in fungi for efficient genetic transformation.     

Study on the electro-transformation conditions of improving transformation efficiency for Bacillus subtilis

Aims: To optimize the transformation conditions and improve the transformation efficiency of Bacillus subtilis WB800 and DB104. Methods and Results: Trehalose, which could decrease the damage of electric shock to the cells, was added to the electroporation medium containing sorbitol and mannitol. The factors affecting the transformation efficiency, such as the growth phase of bacteria, cell concentration, electric field strength and plasmid variety, were examined and improved. The new method increased the transformation efficiency of B. subtilis by nearly 100‐fold compared with the conventional one. Conclusions: With the optimized method, the transformation efficiency came up to 3·64 × 10⁵ transformants μg^?1 DNA for WB800, and 2·10 × 10⁵ transformants μg^?1 DNA for DB104. Significance and Impact of the Study: This improvement in transformation efficiency will be largely attributed to the research of expression of exogenous genes in B. subtilis, gene library construction for directed evolution and transformation of wild‐type B. subtilis strains.     

Characterization of DNA uptake by the cyanobacterium Anacystis nidulans

Summary The binding and uptake of nick-translated ³²P-labeled pBR322 by Anacystis nidulans 6301 have been characterized. Both processes were considerably enhanced in permeaplasts compared to cells. The breakdown of labeled DNA was not correlated with binding or uptake by permeaplasts or cells. Uptake of DNA by permeaplasts was unaffected by: Mg²⁺ or Ca²⁺, light, or inhibitors of photophosphorylation such as valinomycin or gramicidin D in the presence or absence of NH₄Cl. ATP at 2.5–10 mM inhibited both binding and uptake of labeled DNA by permeaplasts of A. nidulans whereas the ATP analog adenyl-5-yl imido-diphosphate was non-inhibitory in the same concentration range. In contrast to transformation of A. nidulans 6301 cells to ampicillin-resistance by pBR322, transformation to kanamycin-resistance by the plasmid pHUB4 was considerably enhanced in the dark. The transformation efficiency for permeaplasts by the plasmid pCH1 was 59% and 8% in the dark and light, respectively, whereas transformation of permeaplasts by pBR322 at an efficiency of 16% was absolutely light-dependent.     

An efficient transformation method for <Emphasis Type="Italic">Bacillus subtilis</Emphasis> DB104

Bacillus subtilis strains are used for extracellular expression of enzymes (i.e., proteases, lipases, and cellulases) which are often engineered by directed evolution for industrial applications. B. subtilis DB104 represents an attractive directed evolution host since it has a low proteolytic activity and efficient secretion. B. subtilis DB104 is hampered like many other Bacillus strains by insufficient transformation efficiencies (≤10³ transformants/μg DNA). After investigating five physical and chemical transformation protocols, a novel natural competent transformation protocol was established for B. subtilis DB104 by optimizing growth conditions and histidine concentration during competence development, implementing an additional incubation step in the competence development phase and a recovery step during the transformation procedure. In addition, the influence of the amount and size of the transformed plasmid DNA on transformation efficiency was investigated. The natural competence protocol is “easy” in handling and allows for the first time to generate large libraries (1.5 × 10⁵ transformants/μg plasmid DNA) in B. subtilis DB104 without requiring microgram amounts of DNA.     

High efficiency electrotransformation of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> spp. <Emphasis Type="Italic">lactis</Emphasis>cells pretreated with lithium acetate and dithiothreitol

Background  

A goal for the food industry has always been to improve strains of Lactococcus lactis and stabilize beneficial traits. Genetic engineering is used extensively for manipulating this lactic acid bacterium, while electropolation is the most widely used technique for introducing foreign DNA into cells. The efficiency of electrotransformation depends on the level of electropermealization and pretreatment with chemicals which alter cell wall permeability, resulting in improved transformation efficiencies is rather common practice in bacteria as in yeasts and fungi. In the present study, treatment with lithium acetate (LiAc) and dithiothreitol (DTT) in various combinations was applied to L. lactis spp. lactis cells of the early-log phase prior to electroporation with plasmid pTRKH3 (a 7.8 kb shuttle vector, suitable for cloning into L. lactis). Two strains of L. lactis spp. lactis were used, L. lactis spp. lactis LM0230 and ATCC 11454. To the best of our knowledge these agents have never been used before with L. lactis or other bacteria.     

Improved Transformation Method for Acetobacter with Plasmid DNA

An improved method for transformation of derivative strains of A. aceti subsp. aceti No. 1023 with plasmid DNA has been developed. Addition of polyethylene glycol or dimethylsulfoxide increased the transformation efficiency by a factor of about ten. In the presence of PEG 4,000, various transformation conditions were examined. Cells were also made transformation competent by treatment with other divalent cations than Ca²⁺ . The pH of the buffer did not affect the efficiency significantly. The growth phase influenced the efficiency. Mutants showing high competence were derived by treatment with N-methyl-N′-nitro-N-nitrosoguanidine. By the improved method using the highly transformable mutants, a transformation efficiency of approximately 105 transformants per γg plasmid DNA was achieved.     

PEG-mediated expression of GUS and CAT genes in protoplasts from embryogenic suspension cultures of Picea glauca

ß-Glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) were used as reporter proteins in protoplasts from embryogenic suspension cultures of Picea glauca (Moench) Voss (white spruce). Plasmid DNA enclosing chimeric GUS and CAT constructs, using the cauliflower mosaic virus 35S promoter, was introduced into Picea glauca protoplasts using polyethylene glycol (PEG). Transient expression was detected 12 to 40 h after PEG-mediated DNA delivery. Dose-response curves using covalently closed circular plasmid DNA, in the absence of carrier DNA, have been obtained for each of these reporter genes. Linearized plasmid DNA gave lower levels of expression than covalently closed circular plasmid DNA when assayed 40 h after PEG-mediated DNA transfer. The use of carrier DNA (herring sperm DNA), in combination with covalently closed circular plasmid DNA, increased the level of expression of GUS by about 50%. CAT expression was enhanced if PEG-mediated delivery was performed on ice rather than at room temperature. The highest level of expression for CAT, and the lowest signal-to-noise ratio, was found 24 h after PEG-mediated DNA transfer. Both GUS and CAT provided results that were quantifiable and can therefore be used as reporter genes in Picea glauca.Abbreviations CAT chloramphenicol acetyl transferase - GUS ß-glucuronidase - CaMV cauliflower mosaic virus - NOS nopaline synthase - CCC covalently closed circular DNA - L linear DNA - PEG polyethylene glycol - HS herring sperm DNA - P protoplasts - PCM protoplast culture medium - MES morpholinoethane-sulfonic acid - Cm chloramphenicol - Ac acetylated - MUG 4-methyl umbelliferyl ß-D-glucuronide - TLC thin layer chromatography     

Germ line transformation of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>, using the transposable element <Emphasis Type="Italic">Minos</Emphasis>

We investigated the use of Minos as a vector for transgenesis in the silkworm, Bombyx mori. We first constructed a vector plasmid with the green fluorescent protein (GFP) gene fused with the silkworm cytoplasmic actin gene (A3) promoter, and a helper plasmid with the Minos transposase gene controlled by the same A3 promoter. Injection of the vector and helper plasmid DNA into silkworm eggs produced transgenic animals in the following generation. The efficiency of transgenic silkworm production using this method was much lower than that obtained using piggyBac-mediated germ line transformation. However, >40-fold increase in the efficiency of producing transgenic silkworms was obtained using an in vitro synthesized source of Minos transposase mRNA. We conclude that the Minos transposon is a useful vector for construction of transgenic silkworms, particularly when in vitro synthesized mRNA is used. This is the first report showing that Minos can be used as a vector for germ-line transformation in lepidopteran insects.     

Restoration of shooty morphology of a nontumorous mutant of Nicotiana glauca x N. langsdorffii by cytokinin and the isopentenyltransferase gene

The shooty morphology of a nontumorous amphidiploid mutant of Nicotiana glauca Grah. x N. langsdorffii Weinm. was restored by cytokinins, whether exogenously applied or endogenously produced by transformation of the mutant with a transfer DNA (T-DNA) cytokinin-biosynthesis gene (isopentenyltransferase; ipt). Auxins alone did not confer this effect. Similar transformation was not achieved for the parental species. In the case of transformation with the ipt gene, selection of the transformed tissues was based on its hormone-independent growth in the presence of the antibiotic kanamycin. Transformed tissues exhibited a shooty morphology, indistinguishable from that of wildtype genetic tumors N. glauca x N. langsdorffii. This altered phenotype was caused by the presence and constitutive expression of the ipt gene. The insertion and expression of this gene in transformed tissues was confirmed by using the polymerase chain reaction (PCR) technique as well as conventional molecular hybridization analysis. Expression of the ipt gene led to an elevated level of cytokinin in the transformed mutant tissues. This evidence supports the notion that genetic tumors are caused, at least in part, by elevated levels of cytokinin in interspecific hybrids.     

Shorter T-DNA or additional virulence genes improve Agrobactrium-mediated transformation

The effect of additional virulence (vir) genes and size of plasmid T-DNA in Agrobacterium tumefa- ciens was investigated for their impact on transformation efficiency. Transformation efficiency in tobacco, cotton, and rice was increased when the T-DNA was 4.3 kb compared to 8.4 kb in size. However, when additional virG, virGN54D,virE, or virE/virG plasmids were included with the 8.4-kb T-DNA, transformation frequencies in all cases were increased over that of the shorter T-DNA without additional vir plasmids. The use of virE, virG or virGN54D copies enhanced transformation efficiency; however, the most significant increase of transformation efficiency in all three plant species was observed when the virE/virG plasmid was used for infection. The virE/virG plasmid dramatically enhanced the efficiency of Agrobacterium-mediated gene transfer; moreover, this plasmid appears to have broad efficiency since it was consistently effective on two different dicotyledon species as well as a monocotyledon species. Received: 8 February 2000 / Accepted: 21 March 2000     

Efficient transformation of<Emphasis Type="Italic"> Medicago truncatula</Emphasis> cv. Jemalong using the hypervirulent<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis> strain AGL1

The efficiency of Agrobacterium tumefaciens transformation of the model legume Medicago truncatula cv. Jemalong (genotype 2HA) was evaluated for strains LBA 4404, C58pMP90, C58pGV2260 and AGL1. Binary vectors carrying promoter-gus/gfp reporter gene fusions and the nptII gene as selectable marker were used for plant in vitro transformation/regeneration. The highest transformation efficiency was obtained with the disarmed hypervirulent strain AGL1 (Ti plasmid TiBo542), for which the percentage of explants forming kanamycin (Km)-resistant calli was double that obtained with each of the other three strains. In addition, we were able to reduce the time necessary for plant regeneration using AGL1, with 24% of the explants generating Km-resistant transgenic plantlets within only 4–5 months of culture. Transgene expression in planta was analysed and found to be conserved in the T₁ descendents.Communicated by R.J. Rose     

A colony‐to‐lawn method for efficient transformation of Escherichia coli

Aims: To develop a fast, convenient, inexpensive and efficient Escherichia coli transformation method for changing hosts of plasmids, which can also facilitate the selection of positive clones after DNA ligation and transformation. Methods and Results: A single fresh colony from plasmid‐containing donor strain is picked up and suspended in 75% ethanol. Cells are pelleted and resuspended in CaCl₂ solution and lysed by repetitive freeze–thaw cycles to obtain plasmid‐containing cell lysate. The E. coli recipient cells are scraped from the lawn of LB plate and directly suspended in the plasmid‐containing cell lysate for transformation. Additionally, a process based on colony‐to‐lawn transformation and protein expression was designed and conveniently used to screen positive clones after DNA ligation and transformation. Conclusions: With this method, a single colony from plasmid‐containing donor strain can be directly used to transform recipient cells scraped from lawn of LB plate. Additionally, in combination with this method, screening of positive clones after DNA ligation and transformation can be convenient and time‐saving. Significance and Impact of the Study: Compared with current methods, this procedure saves the steps of plasmid extraction and competent cell preparation. Therefore, the method should be highly valuable especially for high‐throughput changing hosts of plasmids during mutant library creation.     

High-efficiency and stable genetic transformation of pear (<Emphasis Type="Italic">Pyrus communis</Emphasis> L.) leaf segments and regeneration of transgenic plants

Pear (Pyrus communis L.) is a nutrient-dense fruit with strong consumer demand and high commercial value. However, most cultivated pear varieties are often susceptible to diseases caused by fungi, bacteria, and viruses. The purpose of the present study was to establish an efficient genetic transformation and regeneration protocol, paving the way for genetic engineering of pear cultivars with enhanced disease resistance. Major factors that influence transformation and regeneration were examined and optimal conditions were established for efficient transformation from leaf explants of ‘Old Home’, a valuable pear interstem and rootstock. High transformation efficiency was achieved largely due to an improved infection/transformation induction strategy. Co-cultivation of Agrobacterium cells and leaf segments on a liquid induction medium yielded a fivefold increase in transformation frequency. Southern hybridization analysis revealed presence of reporter gene uidA in the genomic DNA samples from independent transgenic plants, confirming the integration of the transgene in recipient pear genomes. The stability of T-DNA integration was evaluated by the consistent presence of the Km selectable marker and the expression pattern of the introduced reporter gene uidA was analyzed by GUS histochemical assay.