Background
Using synthetic biology methods, the Escherichia coli K-12 genome was reduced by making a series of planned, precise deletions. The multiple-deletion series (MDS™) strains (1), with genome reduction of up to 15%, were designed by identifying non-essential genes and sequences for elimination, including recombinogenic or mobile DNA and cryptic virulence genes, while preserving robust growth and protein production. Genome reduction also led to unanticipated beneficial properties, including high electroporation efficiency and accurate propagation of recombinant genes and plasmids that are unstable in other strains. Subsequent deletions and introduction of useful alleles produce strains suitable for many molecular biology applications. Recently, Scarab has built on the MDS™42 foundation strain, by creating the MDS™42 Meta ΔrecA strain. It improves the already high density fermentation of the MDS™42 strain. The MDS™42 Meta ΔrecA strain’s optimized metabolism permits fermentation ≥OD300 in minimal media in ~24 hrs without glucose spike or cell lysis resulting in >40 g/L of a test protein at the 10 liter scale. It produced ~700 mg/L of pGWIZ GFP test plasmid in minimal media at the 10 liter scale without a temperature shift.
Figures
Figure 1. High Plasmid Yield without a Temperature Shift using Minimal Media. The MDS™42 Meta ΔrecA strain produced ~700 mg/L of pGWIZ GFP test plasmid in 10 liter scale fed batch fermentation without a temperature shift using Scarab’s ultra minimal media. Figure 2: Multiple Deletion Strains tolerate "deleterious” genes. A chimeric gene composed of VP60 of rabbit hemorrhagic disease virus fused to the B subunit of cholera toxin (CTX) was very unstable in E. coli. Individually, both genes were stable in E. coli HB101, C600 and DH10B, but pCTXVP60 carrying the fusion gene in the same hosts did not produce fusion protein and was recovered in low yields. All recovered plasmids contained mutations in the CTXVP60 open reading frame, virtually all resulting from IS insertions. In contrast, the recombinant plasmid was completely stable in MDS™; normal yields of plasmid DNA were obtained. Representative restriction patterns of pCTXVP60. (A) Plasmid DNA from MDS™42 was transformed and propagated in the indicated host, then digested with NcoI and EcoRI. A representative of each restriction pattern was purified and sequenced. M, molecular weight marker, 1 kbp ladder; 1, MDS™41, no insertion; 2, MDS™42, no insertion; 3, DH10B, IS10 insertion; 4, DH10B, IS10 insertion/deletion; 5, C600, IS5 insertion; 6, C600, IS1 insertion; 7, C600, IS1 insertion. (B) Relative position of the IS element insertion sites in the CTXVP60 reading frame determined for the five examples presented. Figure 3: Plasmid stability in different host strains. Left: during four subcultures of pT-ITR, a plasmid with viral LTR segments; Lane 0, isolated plasmid DNA before subculture, lanes 1-4, successive subcultures. Plasmid DNA was digested with restriction enzymes and analyzed by agarose gel electrophoresis. KpnI cuts the plasmid at a single site, but in MG1655 two bands indicate a deletion in the plasmid. MscI cuts at two locations, but in MG1655 a third intermediate band confirms that the plasmid is deleted. Right: Stability of four variants of a Lentiviral expression plasmid in MDS™42 ΔrecA and Stbl3™ (Life Technologies), showing the proportion of transformants containing intact plasmids (Table 2 BioTechniques 43:466-470 (October 2007)(2).
Specifications
Kit Components MDS™42 Meta ΔrecA Electrocompetent Cell Kit pUC19 Control DNA (10 pg/µl) SOC Medium Genotypes MG1655 multiple-deletion strain (1) relA* Δrph ΔarpA ΔiclR ilvG+ ΔrecA(1819). Quality Control Transformation efficiency is tested using pUC19 Control DNA, in duplicate. Transformed cells are plated onto LB plates containing 50 µg/ml carbenicillin. Transformation efficiency is > 5 x 109 cfu/µg DNA. Storage Conditions Store components at –80°C. Do not store cells in liquid nitrogen.
Related Products
White Glove IS Detection Kit
Support
Product Manuals MDS™42 Meta ΔrecA Electrocompetent Cell Kit Papers
- Pósfai G, et al., (2006) Emergent properties of reduced-genome Escherichia coli. Science 312:1044-6.
- Chacko S. Chakiath, CS & Esposito, D (2007): Improved recombinational stability of lentiviral expression vectors using reduced-genome Escherichia coli. BioTechniques 43:466-470.
Patents & Disclaimers
Products are sold for non-commercial use only, under Scarab Genomics limited use label license: Limited Label Use.Scarab is providing you with this Material subject to the non-transferable right to use the subject amount of the Material for your research at your academic institution. The Recipient agrees not to sell or otherwise transfer this Material, or anything derived or produced from the Material to a third party. NO RIGHTS ARE PROVIDED TO USE THE MATERIAL OR ANYTHING DERIVED OR PRODUCED FROM THE MATERIAL FOR COMMERCIAL PURPOSES. If the Recipient makes any changes to the chromosome of the Material that results in an invention in breach of this limited license, then Scarab will have a worldwide, exclusive, royalty-free license to such invention whether patentable or not. If the Recipient is not willing to accept the terms of this limited license, Scarab is willing to accept return of this product with a full refund, minus shipping and handling costs. For information on obtaining a license to this Material for purposes other than research, please contact Scarab’s Licensing Department. Scarab Genomics’ technology is covered by U.S. Pat. No. 6,989,265 and related foreign applications. Clean Genome® is a registered trademark of Scarab Genomics, LLC.
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步骤/方法
将双手放于下巴处,用手指的指腹分别以转圈的方式向耳部方向按摩,并在耳部下方轻轻按压。反复按摩并持续2分钟。由于淋巴是毒素排出的重要渠道,反复按摩此处.有助于琳巴疏通,使毒素顺利排出
双手从下巴处以画圈的方式向嘴角按摩,并在嘴角处轻轻按揉,反复按摩并持续2分钟。下巴常常会堆积很多的毒素,也是比较容易生出粉刺和痘痘的地方。经常对下巴进行按摩可以有助于毒素的排出
.双手无名指指腹放在眼角部位沿着鼻子两侧轻轻从上住下按摩,反复按摩并持续2分钟,此动作可以促进鼻子子部位的血液循环,有助于顺利排出毒素
手指并拢,将双手指腹放在眼睛下方,慢慢向两耳处轻轻按摩,然后再从鼻子中间两侧的脸颊处向耳朵方向轻轻按摩,反复按摩3分钟。此按摩可以加速脸部的血液循环,促进脸部细胞的活化,有助于排除毒素
将双手分别放于嘴角处,然后以画圈的方式向着耳朵的方向轻轻按揉。由于嘴角到耳朵的沿线分布普很多的穴位,经常按摩可以促进细胞的新陈代谢,排出毒素,还可以减缓衰老
现在急需使用这个机器,补试验,不知道哪位能够提供重庆的Seahorse机器信息。非常感谢!
Seahorse细胞代谢分析
美国海马细胞能量代谢实时测定仪/生物能量代谢测定仪XF(SeahorseXFExtracellularFluxAnalyzers)——2009年全球创新技术产品Top10!
美国海马生物科学利用细胞外流量(ExtracellularFlux,XF)检测专利技术,发明了业界第一款海马细胞能量代谢实时测定仪/生物能量代谢测定仪XF24/96,是进行细胞代谢分析、氧呼吸测定、药物代谢分析、线粒体有氧代谢和糖酵解等功能的最佳分析工具。
美国海马细胞能量代谢实时测定仪/生物能量代谢测定仪XF24通过特殊的细胞培养微孔板设计,在测量时临时形成的约5ul微环境中,利用无创的专利光学传感器同步地实时探测溶解氧(OCR)和pH值变化,从而快速了解细胞内两大能量转换途径(线粒体的有氧代谢和糖酵解)的能量代谢状态。在使用XF24的检测过程中,研究人员可以通过预设程序控制在特定时间向待测细胞的培养基中添加多达四种药物,以便研究不同药物对细胞新陈代谢的影响,理解细胞的生物能量变化,快速解析细胞或组织的基础代谢率、ATP转换、膜的完整性、极限呼吸率、线粒体功能,产生氧自由基及超氧化物等有毒物的情况,省时省力,实验数据更科学,更具有说服力。
康乃尔大学应用暨工程物理学(AppliedEngineeringPhysics)副教授KarlA.Kasischke等人成功利用多光子显微技术发现脑部神经细胞(neurons)和星状细胞(astrocytes)之间的如何地交互作用来燃烧氧气和葡萄糖进行糖解作用(glycosis)﹐以达到脑部特别能量的需求。其结果已发表于今年七月的《科学》(Science)杂志上。
该研究团队表示﹐他们根据大脑代谢的辅?烟碱醯胺腺嘌呤双核甘酸(NADH,nicotinamideadeninedinucleotide)两种不同能源状态的影像﹐将最具争议性脑细胞能量代谢的星状细胞—神经元乳酸穿梭(theastrocyte-neuronlactateshuttle)假设作确认与再定义。
KarlA.Kasischke说道﹐在过去十年当中﹐科学家们激烈争议讨论﹐被激活的大脑究竟是进行有氧代谢把葡萄糖彻底分解成水?还是进行无氧状态的糖解作用产生乳酸(lactate)?他表示﹐他们的研究已经发现星状细胞糖解作用伴随着神经活化引发神经性氧化代谢(NeuronalOxidativeMetabolism)将这两种目前对立的说法产生一致性并造成两派双赢的局面。由于他们所使用的多光子显微镜可以让NADH产生内生性荧光影像﹐显示出脑神经内早期氧化代谢终究是持续的﹐并且在约10秒后让星状细胞—神经元乳酸穿梭(theastrocyte-neuronlactateshuttle)作脑细胞晚期的活化作用。神经细胞甚至在休息的时候是不断代谢葡萄糖﹐并且当讯号开始穿越神经细胞时﹐代谢葡萄糖的现象会持续表达﹐然后星状细胞会将代谢葡萄糖所得到的乳酸﹐提供出来做为燃料。
目前医师所使用的脑神经影像技术﹐例如功能性磁共振影像(fMRI,functionalmagneticresonanceimaging)和正子造影系统(PET;positronemissiontomography)虽然可分别探测血流和血氧变化﹐提供医师了解大脑功能变化﹐但是在时间和空间的分辨率却无法满足研究人员的需求。而相较之下﹐多光子显微技术却能提供中枢神经系统(CNS;centralnervoussystem)高分辨率﹐3D立体的组织影像﹐强力地帮助研究人员探讨脑细胞代谢途径。
这场十多年来的争论﹐看来各持己见的双方都没有输。不过﹐最重要的意义是﹐多光子显微技术足以提供大脑代谢等研究功能性方面的应用﹐并且提供给医师较佳的方式来观察中风或阿兹海默症等脑部损害。
全文链接:http://www.sciencemag.org/cgi/reprint/305/5680/50.pdf
酶的作用是催化剂,促进或抑制反应的进行.加热主要是通过升高温度加快反应速率,无机催化剂和酶的原理相同,都是通过降低反应的活化能加快反应速率
解析:1.细胞质基质(也叫胞质溶胶)是指除细胞器外细胞质的其余部分。细胞质基质是活细胞进行新陈代谢的主要场所。
2.细胞新陈代谢的次要场所是:细胞核、线粒体基质、叶绿体基质等基质。
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