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 LowMut strain. It improves the already low mutation rate of the MDS™42 foundation strain. The MDS™42 Meta LowMut strain has been engineered to greatly reduce error-prone repair, which reduces the mutation rate to almost zero, even under the most stressful conditions, thus ensuring the most accurate replication of your plasmid. In addition, its metabolism has been optimized to enable ULTRA high density fermentation ~300 OD600 in minimal media at the 10 liter scale, which in turn enables ULTRA high biotherapeutic yields, protein or plasmid.
Figures
Figure 1. MDS™42 Meta LowMut has the Lowest Mutation Rate Under Stress. Mutation rates of various strains under unstressed and stressful conditions were determined. Stress conditions include overproduction of GFP, overproduction of a toxic peptide from pSG-ORF238 and treatment with mitomycin-C. All measurements were made using the cycA fluctuation assay, error bars represent 95% confidence intervals for the average of 3 independent measurements. BL21(DE3) failed to grow in the presence of 0.1 μg/ml mitomycin-C. ANOVA revealed a significance of p < 0.0001. Pairwise t-tests were conducted for each strain under a given condition compared to the corresponding MDS™42_lowmut strain. Figure 2: Non-Expressing Plasmid Mutations Accumulate rapidly in BL21(DE3), When a Toxic Methyltransferase is Overproduced. SinI methyltransferase was expressed from pSin32. Plasmids were isolated at various intervals and screened (by transformation in McrBC+ and McrBC- hosts) for mutations resulting in loss of function of the enzyme. Error bars represent 95% confidence intervals for the average of 3 independent measurements of mutant plasmid ratios. ANOVA revealed a significance of p < 0.005. Pairwise t-tests of each MDS™42_lowmut_mcrBC sample were done with the corresponding MDS™42 mcrBC and BL21(DE3) mcrBC sample, respectively. Starting from 10 hours, all MDS™42_lowmut_mcrBC samples differed significantly from the MDS™42 mcrBC (p < 0.01) or BL21(DE3) mcrBC (p < 0.005) samples. Figure 3: 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 4: 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 LowMut Electrocompetent Cells pUC19 Control DNA (10 pg/µl) SOC Medium Genotypes MG1655 multiple-deletion strain (1) relA* Δrph ΔarpA ΔiclR ilvG+ ΔdinB ΔpolB ΔumuDC (2). 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 LowMut Electrocompetent Cell Kit Papers
- Pósfai G, et al., (2006) Emergent properties of reduced-genome Escherichia coli. Science 312:1044-6.
- Csörgő et al. (2012) Low-Mutation-Rate, Reduced-Genome Escherichia coli an Improved Host for Faithful Maintenance of Engineered Genetic Constructs Microbial Cell Factories, 11:11.
- 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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研究人员将多效生长因子注入骨髓生长因受到辐射而被抑制的实验鼠体内,后者的骨髓干细胞生长速度与未注射多效生长因子的实验鼠相比提高了10倍。在实验室培养皿中,多效生长因子还被确认可促进人类脐带血干细胞的生长。研究人员还证实,多效生长因子不会导致实验鼠出现癌变。
研究人员说,这项成果将来有望使更广泛的人群受益于脐带血移植,更重要的是,对正在接受化疗或放疗的患者而言,利用多效生长因子进行的治疗或许具有加速患者血液和免疫系统恢复的潜力。
【原文见附件】
ellsNatureMedicineNaturePublishingGroup.pdf(1020.57k)
CristinaAlberini教授的个人主页:http://www.mountsinai.org/profiles/cristina-alberini
全文下载:
nature09667.pdf(558.65k)
结肠炎是一种影响肠道的严重疾病。对结肠炎病人而言,免疫系统抵抗人体自身的肠道细菌,从而导致炎症产生。为了抵抗这种炎症,科学家们已着重关注一种被称作IL-10的化学信号分子。IL-10是一种抗炎性细胞因子。尽管已知IL-10在控制炎症和阻止肠炎中发挥着至关重要的作用,但是仍不清楚的是,它是如何做到这一点的。
在一项新的研究中,来自美国耶鲁大学医学院和哈佛医学院的研究人员以缺乏这种IL-10信号的小鼠和病人为实验对象,研究了这种炎性反应。他们发现IL-10的作用机制是阻断巨噬细胞(作为这种炎性反应的一部分)的代谢。具体而言,他们发现IL-10抑制脂多糖诱导的葡萄糖摄取和糖酵解,促进氧化磷酸化。再者,他们还证实IL-10通过诱导一种被称作DDIT4的mTOR抑制剂产生来抑制mTOR活性。相关研究结果发表在2017年5月5日的Science期刊上,论文标题为“Anti-inflammatoryeffectofIL-10mediatedbymetabolicreprogrammingofmacrophages”。论文通信作者为耶鲁大学医学院免疫学系研究员RuslanMedzhitov。
这些研究人员也注意到IL-10通过促进线粒体自噬(mitophagy)来清除受损的线粒体。这种细胞损伤的堆积会促进炎症产生。线粒体是细胞内的能量工厂。受损线粒体的特征是较低的膜电势和高水平的活性氧。在结肠炎模式小鼠和炎症性肠病患者体内,当IL-10信号缺乏时,巨噬细胞内堆积着受损的线粒体,这会导致NLRP3炎性体异常激活和IL-1β产生。
这些发现加深了对炎症中的一种关键过程的理解,而且可能导致人们开发出靶向结肠炎中的这个通路的疗法。它也可能在阻止或治疗因细胞损伤导致的经常是在衰老时发生的退行性疾病中发挥着重要作用。
原始出处:
W.K.EddieIp,NamikoHoshi,DrorS.Shouvaletal.Anti-inflammatoryeffectofIL-10mediatedbymetabolicreprogrammingofmacrophages.Science,05May2017,356(6337):513-519,doi:10.1126/science.aal3535
AAgnieszkaM.Kabat,EdwardJ.Pearce.Inflammationbywayofmacrophagemetabolism.Science,05May2017,356(6337):488-489,doi:10.1126/science.aan2691
(1)绝大多数细胞因子为分子量小于25kDa的糖蛋白,分子量低者如IL-8仅8kDa。多数细胞因子以单体形式存在,少数细胞因子如IL-5、IL-12、M-CSF和TGF-β等以双体形式发挥生物学作用。大多数编码细胞因子的基因为单拷贝基因(IFN-α除外),并由4-5个外显子和3-4个内含子组成。
(2)主要与调节机体的免疫应答、造血功能和炎症反应有关。
(3)通常以旁分泌(paracrine)或自分泌(autocrine)形式作用于附近细胞或细胞因子产生细胞本身。在生理状态下,绝大多数细胞因子只有产生的局部起作用。
(4)高效能作用,一般在pM(10-12M)水平即有明显的生物学作用。
(5)存在于细胞表面的相应高亲和性受体数量不多,在10-10000/每个细胞。细胞因子受体的研究进展相当迅速,根据细胞因子受体基因DNA序列以及受体胞膜外区氨基酸序列、同源性和结构,可分为四个类型:免疫球蛋白超家族、造血因子受体超家族、神经生长因子受体超家族和趋化因子受体。
(6)多种细胞产生,一种IL可由许多种不同的细胞在不同条件下产生,如IL-1除单核细胞、巨噬细胞或巨噬细胞系产生外,B细胞、NK细胞、成纤维细胞、内皮细胞、表皮细胞等在某些条件下均可合成和分泌IL-1。
(7)多重的调节作用(multipleregulatoryaction),细胞因子不同的调节作用与其本身浓度、作用靶细胞的类型以及同时存在的其它细胞因子种类有关。有时动物种属不一,相同的细胞因子的生物学作用可有较大的差异,如人IL-5主要作用于嗜酸性粒细胞,而鼠IL-5还可作用于B细胞。
(8)重叠的免疫调节作用(overlappingregulatoryaction),如IL-2、IL-4、IL-9和IL-12都能维持和促进T淋巴细胞的增殖。
(9)以网络形式发挥作用,细胞因子的网络作用主要是通过以下三种方式:(1)一种细胞因子诱导或抑制另一种细胞因子的产生,如IL-1和TGF-β分别促进或抑制T细胞IL-2的产生;(2)调节同一种细胞因子受体的表达,如高剂量IL-2可诱导NK细胞表达高亲和力IL-2受体;(3)诱导或抑制其它细胞因子受体的表达,如TGF-β可降低T细胞IL-2受体的数量,而IL-6和IFN-γ可促进T细胞IL-2受体的表达。
(10)与激素、神经肽、神经递质共同组成了细胞间信号分子系统。
(11)自限性分泌。向左转|向右转
干扰素(Interferon,IFN),是由英国科学家Isaacs于1957年利用鸡胚绒毛尿囊膜研究流感病毒干扰现象时首先发现的,是一种细胞因子,具有抑制细胞分裂、调节免疫、抗病毒、抗肿瘤等多种作用。其本质是蛋白质,类型可分为α、β、γ、ω等几种。IFN能诱导细胞对病毒感染产生抗性,它通过干扰病毒基因转录或病毒蛋白组分的翻译,从而阻止或限制病毒感染,是目前最主要的抗病毒感染和抗肿瘤生物制品。
而对于大多数革兰阳性细菌,喹诺酮类药物主要抑制细菌的拓扑异构酶Ⅳ,拓扑异构酶Ⅳ为解链酶,可在DNA复制时将缠绕的子代染色体释放。向左转|向右转