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 ΔrecA strain. It improves the already low mutation rate of the MDS™42 foundation strain. The MDS™42 Meta LowMut ΔrecA 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 ΔrecA 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 ΔrecA Chemically Competent Cells pUC19 Control DNA (10 pg/µl) SOC Medium Genotypes MG1655 multiple-deletion strain (1) relA* Δrph ΔarpA ΔiclR ilvG+ ΔdinB ΔpolB ΔumuDC (2) ΔrecA(1819). Quality Control Transformation efficiency is tested using pUC19 control DNA, performed in duplicate. Transformed cells are plated on LB plates containing 50 μg/ml carbenicillin. Transformation efficiency is ≥1x108 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 ΔrecA Chemically Competent 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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1;变阻脉冲法,光电比色法
2;荧光干涉法
那种的好一点
以前用countstar,一块计数板5个孔,后来实验室又换了biorad的TC20,一块板子两个孔,只能计两个数,一盒板子30片500块,价格越来越贵。这样我一个12孔板计个数就要花百来块钱啊。大家都是这么用的?板子是塑料的,有没有什么可以清洗之后重复利用的方法?
这样的回答可能没分,好和不好都是比较出来的,一群国产的放在一起比总有个性能比较均衡,比较突出性价比好的,
还是没有和说那个厂家的好桂林的呢好像是优利特这个厂家在做,特康的前身呢就是百特,现在的百特呢基本上是在做试剂,这些东西呢。
也搜索的到的,不过答案还是比较接近的,但想知道的是国产那个厂家生产的比较好,而只回答了一半就和介绍了下国产的血球分析仪有那个几个大的厂家,没有和区分那个最好那个第二那个次点,或许一堆鸡蛋里实在挑不出骨头。
具所知呢迈瑞的好点特康实力没有迈瑞好,迈瑞吸引了一批外资在搞研发,桂林的呢好像有个优利特现在出血分析仪,他做的尿分析仪市场的占有还可以,血的反应不是很强烈。
实在是想找个内行的给比较下。
需要测量转染之后细胞某mRNA表达变化。
实验组给予转染,空白组做对照。
做PCR的时候肯定是要求两组的细胞计数是一样的,请问是转染之前还是转染之后对两组进行计数呢?如果样品两边计数不一样,怎么让做实验的时候两边细胞数目变成一样呢?
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