Production of green fluorescent protein by the methylotrophic bacteriumMethylobacterium extorquens
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The production of green fluorescent protein (GFP) in Methylobacterium extorquens was studied by creating four different constructs using pJB3KmD, pRK310 and pVK101 vectors, as well as pLac and soluble methane monooxygenase (sMMO) promoters. Plasmids were introduced into the cells by electroporation. Expression of GFP by selected clones was evaluated by growing cells in complex or defined media. The use of pRK310 as an expression vector containing the lacZ promoter resulted in a 100-fold increase of GFP production when compared to cells containing the pLac-GFP-pJB3KmD construct. Higher production of GFP was observed also in cells containing pLac-GFP-pRK310 and pmmoX-GFP-pVK101 constructs. While the transcriptional regulation of the smmo gene in Methylosinus trichosporium OB3b is known to be copper-dependent, expression of GFP by M. extorquens clones harboring pmmoX-promoters was not strongly controlled by the presence of copper in the medium. The production of GFP was generally constant throughout the growth of M. extorquens carrying the pLac-GFP-pRK310 construct. GFP yields varied between 850 and 1000 microg of GFP g biomass(-1). However, the yield of GFP in cells carrying pmmoX-GFP-pVK101 was somewhat reduced after the mid-exponential phase of growth.Keywords:
Methylobacterium
The mechanism of electroporation has been successfully exploited to deliver gene and drugs into tissue. Electroporation has become an important role to enhance the delivery of genes into tissue. This technique can also be effectively utilized with marginal effect on viability. Electroporation technique can also provide high transfection efficiency. Therefore, there is an increasing interest in electroporation method for delivering gene into tissue. One important area is the delivery of genes into tissues as a therapy. This paper presents the overview of optimal parameters of electroporation for gene and tissue in vivo. This article reviews the obtained results, including the background of electroporation and essential parameters for transfection. This study discusses the evidence based on the success of delivering gene in vivo and effective delivery of drugs in electroporation.
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It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.
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瞄准：在老鼠在调停 electroporation 的基因转移期间在 transgene 表示上调查 Ca2+ 的特定的效果。方法：骨胳的肌肉和皮肤与一个酶记者原生质标志受到在活体内 electroporation，与或没有 Ca2+ 和许多其他的离子。结果：为在活体内 electroporation，在 DNA 解决方案的 10 就 mmol/L Ca2+ 的存在急速地减少了产生 transgene 表达式，到不到 5% 控制值。仅仅 Ca2+ ，不是另外的离子，引起的抑制，和效果不是织物 specific。更令人惊讶地，甚至当 Ca2+ 离子被 electroporation 在 DNA 管理前后交付时，类似的效果仍然被观察。结论：由 electroporation 的在活体内基因转移上的 Ca2+ 的禁止的效果是特定的，即，禁止的效果可能与在 electroporation 和随后的重新封上的事件以后的房间膜性质有关。
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Two endophytic strains of Methylobacterium spp. were used to evaluate biofilm formation on sugarcane roots and on inert wooden sticks. Results show that biofilm formation is variable and that plant surface and possibly root exudates have a role in Methylobacterium spp. host recognition, biofilm formation and successful colonization as endophytes.
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A nif H_ gfp expression vector pMGFP2 was constructed by fusing the 725 bp PCR amplified triple mutated green fluorescent protein ( GFP ) gene ( gfp S65T,V68L,S72A ) fragment to the nif H promoter and its start codon which was from Klebsiella pneumoniae (Schreter) Trevisan M5a1. A kanamycin cassette was inserted into Pst Ⅰ site of pMGFP2, obtaining the expressing vector pMGFP2.1 which can be used for the studying of nif H_ gfp expression in Enterobacter gergoviae 57_7. It was then transformed into E.gergoviae 57_7 and the effects of NH + 4 and oxygen on the expression of nif H_ gfp in E. gergoviae 57_7 were studied.
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Electroporation is a process in which a controlled electrical pulse is applied to cells, inducing a transient destabilization of the cell membrane. During this time, the cells are highly permeable to exogenous substances in the surrounding media. DNA, proteins, and small molecules are all taken up by cells during electroporation; introduction of DNA into cells is the most common application. Gene transfer by electroporation offers many advantages for analysis of gene expression. The technique is simple, rapid, and reproducible. It is especially suited to suspension cultures and certain cell types that are poorly trans-fected by other means. Because all cells are transfected instantaneously, and essentially simultaneously, it is particularly suited to quantitation of gene transfer. Finally, single-copy, stable transfectants can often be isolated (1). Whereas the basic mechanisms of electroporation are still largely unknown, optimizing the conditions for electroporation of any particular cell type is primarily empirical.
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Gene transfer
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Electroporation-the use of high-voltage electric shocks to introduce DNA into cells-can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternative techniques. This unit describes electroporation of mammalian cells, including ES cells, for the preparation of knock-out, knock-in, and transgenic mice. Protocols are described for the use of electroporation in vivo to perform gene therapy for cancer, as well as for DNA vaccination. © 2017 by John Wiley & Sons, Inc.
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Two aerobic, pink-pigmented, facultatively methylotrophic bacteria, strains F20T and RXM(T), are described taxonomically. On the basis of their phenotypic and genotypic properties, the isolates are proposed as novel species of the genus Methylobacterium, Methylobacterium suomiense sp. nov. (type strain F20T = VKM B-2238T = NCIMB 13778T) and Methylobacterium lusitanum sp. nov. (type strain RXMT = VKM B-2239T = NCIMB 13779T).
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A pink-pigmented, aerobic, facultatively methylotrophic bacterium, strain BJ001 T , was isolated from internal poplar tissues ( Populus deltoides × nigra DN34) and identified as a member of the genus Methylobacterium . Phylogenetic analyses showed that strain BJ001 T is related to Methylobacterium thiocyanatum , Methylobacterium extorquens , Methylobacterium zatmanii and Methylobacterium rhodesianum . However, strain BJ001 T differed from these species in its carbon-source utilization pattern, particularly its use of methane as the sole source of carbon and energy, an ability that is shared with only one other member of the genus, Methylobacterium organophilum . In addition, strain BJ001 T is the only member of the genus Methylobacterium to be described as an endophyte of poplar trees. On the basis of its physiological, genotypic and ecological properties, the isolate is proposed as a member of a novel species of the genus Methylobacterium , Methylobacterium populi sp. nov. (type strain, BJ001 T =ATCC BAA-705 T =NCIMB 13946 T ).
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It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.
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Primary cell
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