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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由于质粒的不兼容性,拥有同种复制子的质粒不能在同一细胞内稳定共存,经过几代的复制,会质粒丢失,所以并不是任何两个或两个以上质粒都可以在同一细胞内稳定存在,但是可以同时进入.
稳定转染的细胞株,就是转染后质粒可以稳定整合到基因组上不会因细胞分裂而丢失。区别于质粒瞬时转染不能长时间保留质粒在细胞里。
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。
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.
2.将构建完成的载体与慢病毒包装质粒混合,共转染靶细胞
3.收集病毒液
4.用病毒液感染靶细胞
5.用载体上带的抗生素进行筛选,如果没有,可以用无限稀释法
6.获得稳转株
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