High-throughput cloning and expression in recalcitrant bacteria

We developed a generic method for high-throughput cloning in bacteria that are less amenable to conventional DNA manipulations. The method involves ligation-independent cloning in an intermediary Escherichia coli vector, which is rapidly converted via vector-backbone exchange (VBEx) into an organism-specific plasmid ready for high-efficiency transformation. We demonstrated VBEx proof of principle for Lactococcus lactis, but the method can be adapted to all organisms for which plasmids are available.     

毕赤酵母基因操作技术的改进及其在水蛭素表达中的应用

毕赤酵母是日益受到重视的基因工程受体菌,但是目前尚没有一种简便、高效的转化方法,也没有一种稳定、可靠的菌落PCR方法。本文通过在通常筛选重组子的MD培养基中增添1mol/L山梨糖醇使毕赤酵母电穿孔转化效率提高10倍以上。并比较系统地分析了其他影响电穿孔转化效率的因素,如毕赤酵母生长状况即OD600,不同的整合位点(BglⅡ,SacⅠ,SalⅠ,StuⅠ),及不同的毕赤酵母受体菌(GS115和KM71)等。本文描述的转化方法所能达到的转化效率比目前大多数文献报道的效率要高,可使每微克质粒DNA产生的整合到受体菌染色体上的转化子述2800个;另外,我们经过一种冷热处理毕赤酵母菌落的方法使其细胞壁裂解,并改变通常PCR缓冲液的组成成分后,毕赤酵母菌落PCR方法几乎和大肠杆菌的操作一样简便可靠。借助本文所描述的电穿孔转化方法和菌落PCR方法,成功实现了水蛭素在毕赤酵母中的分泌表达,表达量为每毫升培养上清20μg,其生物活性达每毫升培养上清82个国际单位。     

A rapid and efficient method for plasmid transformation of Klebsiella pneumoniae and Escherichia coli

A rapid and efficient method for plasmid transformation of Klebsiella pneumoniae M5a1 and Escherichia coli K12 has been developed. The method, which uses a freeze-thaw cycle in the presence of CaCl2 to facilitate DNA uptake, is substantially more efficient for K. pneumoniae M5a1 than the conventional transformation procedure for E. coli. The simplicity and speed of the method makes it very attractive for routine transformation of K. pneumoniae M5a1 and E. coli K12.     

Plasmid uptake by bacteria: a comparison of methods and efficiencies

The ability to introduce individual molecules of plasmid DNA into cells by transformation has been of central importance to the recent rapid advancement of plasmid biology and to the development of DNA cloning methods. Molecular genetic manipulation of bacteria requires the development of plasmid-mediated transformation systems that include (1) chemical transformation, (2) electro-transformation, (3) biolistic transformation, and (4) sonic transformation, leading to the introduction of exogenous plasmid DNA into bacterial cells. In this review, the manipulation properties and transformation efficiencies of these techniques are described. In addition to these methods, a conceptually novel transformation technique, namely the hydrogel exposure method, was developed. The hydrogel exposure method, based on the Yoshida effect, provides a significant advance over chemical means for transforming many strains of Escherichia coli and a variety of other bacterial species. The new term “tribos transformation” has been proposed for this novel technique. We also determined that, compared to conventional methods, the hydrogel exposure method is a novel and convenient method by which to transform bacteria.     

Biolistic transformation of arbuscular mycorrhizal fungi

Gene transfer systems have proved effective for the transformation of a range of organisms for both fundamental and applied studies. Biolistic transformation is a powerful method for the gene transfer into various organisms and tissues that have proved recalcitrant to more conventional means. For fungi, the biolistic approach is particularly effective where protoplasts are difficult to obtain and/or the organisms are difficult to culture. This is particularly applicable to arbuscular mycorrhizal (AM) fungi, being as they are obligate symbionts that can only be propagated in association with intact plants or root explants. Furthermore, these fungi are aseptate and protoplasts cannot be released. Recent advancements in gene transformation systems have enabled the use of biolistic technology to introduce foreign DNA linked to molecular markers into these fungi. In this review we discuss the development of transformation strategies for AM fungi by biolistics and highlight the areas of this technology which require further development for the stable transformation of these elusive organisms.     

A simple and rapid method for intra- and interspecific transformation of Bacillus subtilis on solid media by DNA in protoplast lysates

A simple method for intra- and interspecific transformation of Bacillus subtilis on solid media has been devised with DNA in protoplast lysates, 0.8% agar, glutamate, and yeast extract. The transformation frequency is 2.3 x 10(3) transformants per microg DNA, 10-20 times higher than that for conventional transformation on solid media. The method can be applicable to transformation in microtiter plates.     

Internal standards for survival: increasing the accuracy for human cell mutation assays

In mutation and cell transformation assays, it has long been recognized that the common practice of using different numbers of cells on dishes with or without selective conditions creates a source of bias in mutant fraction determination. This is simply because colony formation may be enhanced or suppressed at higher initial cell densities, depending on the assay and agent tested. We propose a solution that consists of the inclusion of an experimentally distinguishable population of cells as an internal standard for colony-forming ability at the high cell density required for detection of rare variants. This method is found to be highly satisfactory for use in measuring mutation to 6-thioguanine resistance in a diploid human B lymphoblast line. For treatment with anti-2,3-dihydroxy-1,10b-epoxy-1,2,3-trihydrofluoranthene (FDE), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and 4-nitroquinoline-oxide (4NQO), the calculated induced mutant fractions using the internal-standard method were significantly lower than those calculated using the conventional low-density-plating efficiency method. The results of these experiments and our analysis lead us to conclude that this approach is applicable to all single cell mutation or transformation assays and is a necessary feature of assays in which an accurate knowledge of the fraction of rare variants is required.     

“Virtues” of apartheid

The traditional clinical method, which has served medicine well for over 100 years, had its origins in the integration of physical examination with morbid anatomy in early 19th-century France. Now this method is showing signs of failing to meet some contemporary needs. In particular, there is no means for understanding the inner experience of patients. Previous models of a transformed method have not grappled with the problem of validation. Data on the inner experience of patients are not open to empiric validation in the same way as clinical data. The process of understanding the meaning of an illness is not, therefore, scientific in the conventional sense. There are, none the less, rigorous methods for validating the results of this form of inquiry, notably those of phenomenology. A transformed method should aim to understand the meaning of an illness for the patient as well as provide a clinical diagnosis. The transformation will require a change in the epistemology of medicine and an educational process that encourages reflection and growth of self-knowledge.     

Continuous measurement of gas uptake and elimination in anesthetized patients using an extractable marker gas.

Measurement of pulmonary gas uptake and elimination is often performed, using nitrogen as marker gas to measure gas flow, by applying the Haldane transformation. Because of the inability to measure nitrogen with conventional equipment, measurement is difficult during inhalational anesthesia. A new method is described, which is compatible with any inspired gas mixture, in which fresh gas and exhaust gas flows are measured using carbon dioxide as an extractable marker gas. A system was tested in eight patients undergoing colonic surgery for automated measurement of uptake of oxygen, nitrous oxide, isoflurane, and elimination of carbon dioxide with this method. Its accuracy and precision were compared with simultaneous measurements made with the Haldane transformation and corrected for predicted nitrogen excretion by the lungs. Good agreement was obtained for measurement of uptake or elimination of all gases studied. Mean bias was -0.003 l/min for both oxygen and nitrous oxide uptake, -0.0002 l/min for isoflurane uptake, and 0.003 l/min for carbon dioxide elimination. Limits of agreement lay within 30% of the mean uptake rate for nitrous oxide, within 15% for oxygen, within 10% for isoflurane, and within 5% for carbon dioxide. The extractable marker gas method allows accurate and continuous measurement of gas uptake and elimination in an anesthetic breathing system with any inspired gas mixture.     

Library-independent cloning of genomic fragments adjacent to vector integration sites: isolation of the EB4-PSV gene from a Dictyostelium gene disruption transformant.

We have designed an efficient procedure to clone genomic DNA adjacent to the integration site of transformation vectors. Using this method on a Dictyostelium gene disruption transformant, we have cloned a 5kb fragment which has previously escaped isolation by conventional library screening. Our protocol eliminates recloning of the original vectors which are often integrated in multiple tandem copies (1) but specifically recovers vectors containing genomic fragments adjacent to the integration site. The protocol is useful to isolate flanking fragments in gene disruption transformants to characterize flanking regions which may influence the expression of transformed genes by position effects and to identify sites of insertional mutagenesis.     

Development of an efficient agrobacterium-mediated gene targeting system for rice and analysis of rice knockouts lacking granule-bound starch synthase (Waxy) and β1,2-xylosyltransferase

We have developed a high-frequency method for Agrobacterium-mediated gene targeting by combining an efficient transformation system using rice suspension-cultured calli and a positive/negative selection system. Compared with the conventional transformation system using calli on solid medium, transformation using suspension-cultured calli resulted in a 5- to 10-fold increase in the number of resistant calli per weight of starting material after positive/negative selection. Homologous recombination occurred in about 1.5% of the positive/negative selected calli. To evaluate the efficacy of our method, we show in this report that knockout rice plants containing either a disrupted Waxy (granule-bound starch synthase) or a disrupted Xyl (β1,2-xylosyltransferase) gene can be easily obtained by homologous recombination. Study of gene function using homologous recombination in higher plants can now be considered routine work as a direct result of this technical advance.     

A Built-In Strategy to Mitigate Transgene Spreading from Genetically Modified Corn

Transgene spreading is a major concern in cultivating genetically modified (GM) corn. Cross-pollination may cause the spread of transgenes from GM cornfields to conventional fields. Occasionally, seed lot contamination, volunteers, mixing during sowing, harvest, and trade can also lead to transgene escape. Obviously, new biological confinement technologies are highly desired to mitigate transgene spreading in addition to physical separation and isolation methods. In this study, we report the development of a built-in containment method to mitigate transgene spreading in corn. In this method, an RNAi cassette for suppressing the expression of the nicosulfuron detoxifying enzyme CYP81A9 and an expression cassette for the glyphosate tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene G10 were constructed and transformed into corn via Agrobacterium-mediated transformation. The GM corn plants that were generated were found to be sensitive to nicosulfuron but resistant to glyphosate, which is exactly the opposite of conventional corn. Field tests demonstrated that GM corn plants with silenced CYP81A9 could be killed by applying nicosulfuron at 40 g/ha, which is the recommended dose for weed control in cornfields. This study suggests that this built-in containment method for controlling the spread of corn transgenes is effective and easy to implement.     

An efficient method for the physical mapping of transgenes in barley using in situ hybridization.

The genetic transformation of crops by particle bombardment and Agrobacterium tumefaciens systems have the potential to complement conventional plant breeding programmes. However, before deployment, transgenic plants need to be characterized in detail, and physical mapping is an integral part of this process. Therefore, it is important to have a highly efficient method for transgene detection by fluorescence in situ hybridization (FISH). This study describes a new approach, which provides efficient control of probe length and labelling, both of which play an important role in in situ hybridization of transgenes. The approach is based on reducing the size of the plasmid prior to labelling by nick translation, rather than using the whole or linearized plasmid, or varying the amounts of DNaseI in the nick translation mixture. This provided much more efficient labelling of the probe, which yielded optimal hybridization. minimal fluorescent background, and accurate physical location of the transgene.     

矮牵牛育种研究进展

从常规育种、基因工程育种、体细胞育种和单倍体育种4个方面评述了矮牵牛(Petunia hybrida Vilm.)育种研究进展.国外对矮牵牛育种研究较多,新品种面世推陈出新,国内对其研究则较为薄弱.其常规育种最为成功,不断有新品种推出,基因工程育种也取得了一定成就,已有新品种面世,而体细胞育种及单倍体育种尚无新品种产生,但对这两种方法在育种上应用的可能性和前景作出了一定探索.     

In vivo construction of transgenes in Drosophila

Transgenic flies are generated by transposon-mediated transformation. A drawback of this approach is the size limit of transposable elements. Here, we propose a novel method that allows the extension of transgenes in vivo. This method is based on an incomplete transgene that has been constructed in vitro and integrated into the Drosophila genome by conventional transgenesis. The incomplete transgene contains two short stretches of DNA homologous to the 5'- and 3'-ends of a larger DNA segment of interest. Between the short stretches of homology an I-SceI recognition site is located. Once activated, I-SceI endonuclease introduces a DNA double-strand break, which triggers ectopic recombination between the stretches of homology and the endogenous locus. Through gap repair, the transgene obtains the complete region of interest in vivo. Our results show that this method is effective for copying up to 28 kb of genomic DNA into the transgene, thereby eliminating the technical difficulties associated with the in vitro construction of large transgenes and extending the size limits of current transgenesis protocols. In general, this method may be a useful technique for genetic engineering of eukaryotic model organisms.     

A simple method of solution for a class of bioreaction-diffusion problems

Summary A simple method of solution is highlighted for solving a class of bioreaction-diffusion problems. By virtue of the proposed transformation the original boundary value problem is rendered into an equivalent initial value problem. Results are comparable to conventional methods, and have lower computational time requirements.NCL Communication. No. 5179     

Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings

The functional genomics approach requires systematic analysis of protein subcellular distribution and interaction networks, preferably by optimizing experimental simplicity and physiological significance. Here, we present an efficient in planta transient transformation system that allows single or multiple expression of constructs containing various fluorescent protein tags in Arabidopsis cotyledons. The optimized protocol is based on vacuum infiltration of agrobacteria directly into young Arabidopsis seedlings. We demonstrate that Arabidopsis epidermal cells show a subcellular distribution of reference markers similar to that in tobacco epidermal cells, and can be used for co-localization or bi-molecular fluorescent complementation studies. We then used this new system to investigate the subcellular distribution of enzymes involved in sphingolipid metabolism. In contrast to transformation systems using tobacco epidermal cells or cultured Arabidopsis cells, our system provides the opportunity to take advantage of the extensive collections of mutant and transgenic lines available in Arabidopsis. The fact that this assay uses conventional binary vectors and a conventional Agrobacterium strain, and is compatible with a large variety of fluorescent tags, makes it a versatile tool for construct screening and characterization before stable transformation. Transient expression in Arabidopsis seedlings is thus a fast and simple method that requires minimum handling and potentially allows medium- to high-throughput analyses of fusion proteins harboring fluorescent tags in a whole-plant cellular context.     

矮牵牛育种研究进展

从常规育种、基因工程育种、体细胞育种和单倍体育种4个方面评述了矮牵牛(Petunia hybrida Vilm.)育种研究进展。国外对矮牵牛育种研究较多,新品种面世推陈出新,国内对其研究则较为薄弱。其常规育种最为成功,不断有新品种推出,基因工程育种也取得了一定成就,已有新品种面世,而体细胞育种及单倍体育种尚无新品种产生,但对这两种方法在育种上应用的可能性和前景作出了一定探索。     

Estimation of the axis of a screw motion from noisy data--a new method based on Plücker lines

The problems of estimating the motion and orientation parameters of a body segment from two n point-set patterns are analyzed using the Plücker coordinates of a line (Plücker lines). The aim is to find algorithms less complex than those in conventional use, and thus facilitating more accurate computation of the unknown parameters. All conventional techniques use point transformation to calculate the screw axis. In this paper, we present a novel technique that directly estimates the axis of a screw motion as a Plücker line. The Plücker line can be transformed via the dual-number coordinate transformation matrix. This method is compared with Schwartz and Rozumalski [2005. A new method for estimating joint parameters from motion data. Journal of Biomechanics 38, 107-116] in simulations of random measurement errors and systematic skin movements. Simulation results indicate that the methods based on Plücker lines (Plücker line method) are superior in terms of extremely good results in the determination of the screw axis direction and position as well as a concise derivation of mathematical statements. This investigation yielded practical results, which can be used to locate the axis of a screw motion in a noisy environment. Developing the dual transformation matrix (DTM) from noisy data and determining the screw axis from a given DTM is done in a manner analogous to that for handling simple rotations. A more robust approach to solve for the dual vector associated with DTM is also addressed by using the eigenvector and the singular value decomposition.     

Interspecific transformation of Bacillus subtilis competent cells by chromosomal DNA in lysates of protoplasts of Bacillus amyloliquefaciens

Competent cells of Bacillus subtilis were transformed with chromosomal DNA in lysates of protoplasts of B. subtilis or B. amyloliquefaciens. The interspecific transformation frequency of B. subtilis by cysA in a conserved region was 3.1 x 10(4) transformants per microg DNA, 60 times higher than that for conventional transformation using purified DNA. Increased interspecific transformation frequencies of B. subtilis were also observed for arg-1, lys-1, leuB, aroG, thr-5, hisH, or metC markers outside the conserved region (3.1 x 10 approximately 5.2 x 10(2) transformants per microg DNA). An interspecific cotransformation ratio (33-50%) as high as an intraspecific one (46%) using purified DNA was also detected between cysA and rpsL markers, which are separated by 16 kb on the B. subtilis chromosome. Interspecific double transformation of the cysA-arg-1 or cysA-metC marker was observed, which have not been detected for conventional transformation. The involvement of mutS in the interspecific transformation was not significant.