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.
Figures
Figure 1: 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 2: 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 Electrocompetent Cells | 0.2 mL |
MDS™42 ΔrecA Electrocompetent Cells | 0.2 mL |
MDS™42 ΔrecA Blue Electrocompetent Cells | 0.2 mL |
pUC19 Control DNA (10 pg/µl) | 50 µL |
SOC Medium | 2 x 10 mL |
Related Products
White Glove IS Detection Kit
Support
Product Manuals MDS™42 Combination Package 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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先提取RNA,反转录成cDNA,然后根据目的基因设计PCR引物,通过半定量RT-PCR确定目的基因表达.
正因为检测的是mRNA,所以要先反转录成cDNA才能PCR.
① 在构建载体时,目的基因直接整合到细胞染色体组上,最好不要通过先瞬转在筛选稳定细胞株的这种方法,因为转染效率没有保证
② 高表达载体的构建,哺乳动物表达量一直是它自身的缺点,最好根据高表达载体定向的驯化细胞,提高蛋白表达量
③ 细胞的选择,筛选稳定细胞株我们常用的细胞是CHO,中国仓鼠卵巢细胞,由于CHO具有诸多的优点因此适合用于筛选稳定细胞株,而HEK293细胞则常用于瞬时转染
④ 后期的筛选,双抗预防污染,筛选细胞的时候抗生素浓度一定要做预实验,而且转染的时候不能有抗生素,关于细胞转染 稳定细胞系构建的相关理论
最近因工作需要,欲购进一些生产用的细胞株和瞬转、稳转质粒,大家有什么推荐的品系和公司或网站,谢谢!!!
主要需求:
1.生产或生物制药用的,稳定细胞株如CHO-K1、GS和HEK293/HEK293T,以及与之对应的瞬转、稳转(重组整合)质粒(需详细图谱)。
2.原始或改造细胞株,以及对应质粒,改造细胞株请详述细节,原因,优缺点,安全性等。
3.希望大家积极推荐,实验用、生产用均可,原核、真核都行,网站或品系名称也行,只要大家觉得常用、稳定、安全、高产就行。
4.请有意向的同仁回帖或将详细资料发至我163邮箱:wangqiang23mars@163.com,万分感谢。
然后你得确定,这个细胞株对于你得研究来说不可或缺,具有明确的代表性。
上面两个都没有问题的话,祝你顺利。
如果经费充足的话可以找公司包装病毒,如武汉的普健可以提供各种载体的构建及细胞株构建的技术服务。
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