Recombinant Bacteria for Protein Engineering

There are two convenient forms of genetic material, that can be used as vehicles for introducing the new gene into the bacterium a small circular DNA piece, called a plasmid, or a virus that grows in bacteria. The techniques described below apply to both plasmids and viruses. 

As the second educt (B), the plasmid ONA with complementary sticky ends is prepared separately. In the first step the isolated plasmid DNA is cut open by a special type of enzyme called restriction endonuclease. It scans along the thread of DNA and recognizes short nucleotide sequences, e.g., CTGCAG, which ate cleaved at a specific site, e.g., between A and G. Some 50 of such enzymes are known and many are commercially available. The ends are then again extended witfa he aid of a terminal transferase by a short sequence of identical nucleotides complementary to the sticky ends of educt (A). 

The synthetic and plasmid DNAs are mixed and join their sticky ends spontaneously. They are covalently bound together by DNA ligases, when the resulting hybrid plasmid is inserted into bacterial cells. Dilute calcium chloride solutions render the bacterial membranes permeable and allow the passage of ONA into the cells. 

The yields of DNA intake by the cells are, however, very low. The few bacteria containing the hybrid plasmid need to be sorted out. One technique applies a plasmid which encodes for antibiotic-degrading enzymes, e.g for penicillinase and tetracyclinase, and thus 

The cyclization reactions discussed here either involve the intramolecular reaction of a donor group D with an acceptor group A or a cyclizing dimerization of two molecules with two terminal acceptors and two donors. A polymerization reaction will always compete with cyclization. For macrolides see p. 146 and p. 319 — 329. 

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A final problem for bioinformatics and bioanalytical scientists is the characterization of engineered microorganisms. Whole-cell analysis by mass spectrometry has been used to confirm the introduction of therapeutic genes into adenovirus vectors,100 to confirm the expression of recombinant proteins in bacteria,101,102 and also in vaccinology.103 In the broader case, identification of... 

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