Transfection into E. coli

In a simple procedure for DNA cloning, an autonomously replicating plasmid and insert DNA are cut with a restriction enzyme and then the pieces are annealed and covalently joined by the action of DNA ligase. The resulting recombinant molecules are then transfected into E. coli, where they replicate. When plasmid vectors are used, a population of permeabilized cells is bathed in the plasmid DNA containing the inserted DNA. Because only a small number of cells become transfected by this procedure, a way to se-... 

A CaaX motif (Cys-Val-lle-Met) can be introduced C-terminally into the protein in question by standard techniques. The cDNA obtained can be cloned into an expression vector for E. coli, such as pTrc-99A (Pharmacia), and then transfected into E. coli DH5 a and induced with 5 mM isopropyl-(3-D-thioglactopyranoside (IPTG). The protein is isolated from the bacterial pellet. 

Transform into E. coli cells, prepare minipreps and identify correct clones by digesting with Sail and Hindlll. Correctly ligated clones show two bands of 1.6 and 3.0 kb, plasmids without insert show only the 4.3 kb vector band. M13 forward, Ml3 reverse, and bpA-for primers can be used for sequence confirmation. Prepare Maxipreps and use two independent plasmids of pRMCE-U6-shRNA for stable transfection by RMCE in ES cells. 

Bacteriophage A vectors that accommodate foreign DNA fragments generated by a variety of restriction endonucleases have been constructed. The recombinant DNA molecules that incorporate some of these vectors can be introduced directly into E. coli by transfection. Alternatively, recombinant DNA molecules can be packaged into phage particles and subsequently infected into suitable host cells. 

The DNA or cDNA library is then introduced into a preparation of bacterial host cells. Usually, the first host selected is a laboratory strain of E. coli which has been grown and pretreated with inorganic salts to make uptake of DNA easier. The ability to take up foreign DNA is called competence, cells which have been specially prepared for the purpose are called competent cells. Other methods to transfer DNA into cells include electroporation (application of an external electric field to permeabUize the cell wall), transfection (where a recombinant bacterial virus is used to transfer the DNA to the target cell) or ballistic methods (by using DNA-coated particle projectiles). The last method has been used to introduce foreign DNA into plant cells and mammalian cells. 

Transfect the final expression vector into competent E. coli strain BMH 71-18. 

Before embarking on a particular expression strategy it is useful to have some information regarding the hands-on time required before the first results can be obtained. The fastest turnaround times are achieved with cell lysates, such as the reticulocyte and wheat germ lysates, which only need to be primed with in vitro synthesized RNA (or DNA in the case of a coupled transcription/translation system). These systems potentially provide answers within a few hours but are rarely used because of the small quantities that they yield. Plasmid-based systems used for expression in E. coli, yeast, and in combination with the vaccinia-T7 vector system are relatively fast as well. These strategies require subcloning of the gene(s) of interest into a plasmid vector, which is then transfected into the... 

The used plasmid pEScFv is a eukaryotic expression vector containing an AM single-chain Fv (ScFv) fragment linked with domain B of a protein A of S. aureus. Sp2/0 myeloma cells transfected with pEScFv 2G42D7 secreted ScFv into the culture media. E. coli strain XLl/Blue is transformed with plasmid pEScFv... 

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