E. Coli cells

Because of the double helical nature of DNA molecules, their size can be represented in terms of the numbers of nucleotide base pairs they contain. For example, the E. coli chromosome consists of 4.64 X 10 base pairs (abbreviated bp) or 4.64 X 10 kilobase pairs (kbp). DNA is a threadlike molecule. The diameter of the DNA double helix is only 2 nm, but the length of the DNA molecule forming the E. coli chromosome is over 1.6 X 10 nm (1.6 mm). Because the long dimension of an E. coli cell is only 2000 nm (0.002 mm), its chromosome must be highly folded. Because of their long, threadlike nature, DNA molecules are easily sheared into shorter fragments during isolation procedures, and it is difficult to obtain intact chromosomes even from the simple cells of prokaryotes. 

The first biologically functional chimeric DNA molecules constructed in vitro were assembled from parts of different plasmids in 1973 by Stanley Cohen, Annie Chang, Herbert Boyer, and Robert Helling. These plasmids were used to transform recipient E. coli cells transformation means the uptake and repli-... 

Recombinant DNA technology now verges on the ability to engineer at will the genetic constitution of organisms for desired ends. The commercial production of therapeutic biomolecules in microbial cultures is already established (for example, the production of human insulin in quantity in E. coli cells). Agricultural crops with desired attributes, such as enhanced resistance to her-... 

Proinsulin fusion E. coli cells sulfitolysis SEC Affix UV 61 B p... 

The chromosomes of Escherichia coli and other bacteria are single, double-stranded DNA molecules with a total length of more than 1,000 pm. Relaxed DNA exists as a helical molecule, with one full turn of the helix occurring approximately every 10.4 base pairs. This molecule must undergo several folding and compaction steps to fit into an E. coli cell which is only 1-3 pm long. Despite this enormous compaction, bacterial DNA must be accessible for the bacterial enzymes that catalize DNA replication and transcription... 

An extract from the soluble stromal proteins of purified and intact spinach-leaf chloroplasts was prepared by lysis of the cells in buffer, centrifugation of the suspension of broken cells, and concentration of the supernatant with removal of insoluble material. This extract contained all of the enzymes involved in the condensation of the cyclic moieties of thiamine, thiazole, and pyramine. Thus, the synthesis of thiamine in this extract following the addition of pyramine and putative precursors was a proof that the system had the possibility of building the thiazole. It was found that L-tyrosine was the donor of the C-2 carbon atom of thiazole, as in E. coli. Also, as in E. coli cells, addition of 1 -deoxy-D-f/irco-pen-tulose permitted synthesis of the thiamine structure. The relevant enzymes were localized by gel filtration in a fraction covering the 50- to 350-kDa molecular-mass range. This fraction was able to catalyze the formation of the thiazole moiety of thiamine from 0.1 -mM 1-deoxy-D-t/ireo-pentulose at the rate of 220 pmol per mg of protein per hour, in the presence of ATP and Mg2+. 

The Rieske protein II (SoxF) from Sulfolobus acidocaldarius, which is part, not of a bci or b f complex, but of the SoxM oxidase complex 18), could be expressed in E. coli, both in a full-length form containing the membrane anchor and in truncated water-soluble forms 111). In contrast to the results reported for the Rieske protein from Rhodobacter sphaeroides, the Rieske cluster was more efficiently inserted into the truncated soluble forms of the protein. Incorporation of the cluster was increased threefold when the E. coli cells were subject to a heat shock (42°C for 30 min) before induction of the expression of the Rieske protein, indicating that chaperonins facilitate the correct folding of the soluble form of SoxF. The iron content of the purified soluble SoxF variant was calculated as 1.5 mol Fe/mol protein the cluster showed g values very close to those observed in the SoxM complex and a redox potential of E° = +375 mV 111). 

What could be the signal for the induction of the cold shock proteins It has been observed that shifting E. coli cells from 37 to 5 °C results in an accumulation of 70S monosomes with a concomitant decrease in the number of polysomes [129]. Further, it has been shown that a cold shock response is induced when ribosomal function is inhibited, e.g. by cold-sensitive ribosomal mutations [121] or by certain antibiotics such as chloramphenicol [94]. These data indicate that the physiological signal for the induction of the cold shock response is inhibition of translation caused by the abrupt shift to lower temperature. Then, the cold shock proteins RbfA, CsdA and IF2 associate with the 70S ribosomes to convert the cold-sensitive nontranslatable ribosomes into cold-resistant translatable ribosomes. This in turn results in an increase in cellular protein synthesis and growth of the cells. 

FIGURE 5.3.5 Enhancement of lycopene accumulation in . coZi by over-expression of DXS. Lycopene accumulation (left) is enhanced (right) when E. coli cells carrying a carotenoid pathway gene cassette (+EIB) are further transformed with a dxs gene on a multicopy plasmid (+E1B +dxs). Lycopene hyperaccumulation was demonstrated by Matthews and Wurtzel. ... 

Concerning the mechanism of action of catechins, studies carried out on S. aureus and E. coli cells by Ikigai et al. [72] reported that their bactericidal effect is primarily involved in the damage of bacterial membranes catechins induce a rapid leakage of small molecules entrapped in the intraliposomal space, determining the aggregation of the liposomes. These actions cause damage in the membrane lipid bilayer and cell death (Table 1). 

Kuhn, R. and Wagner, H., Application of free flow electrophoresis to the preparative purification of basic proteins from an E. coli cell extract,... 

Figure 5.10. Protein complementation assay using murine DHFR. The F[l,2] and F[3] fragments are each fused to the homodimerizing GCN4 leucine zipper protein. A. Transformation of both Z-F[l,2] and Z-F[3] constructs results in reconstituted DHFR and growth of E. coh on agar plates containing trimethoprim. B. Transformation of Z-F[l,2] or Z-F[3] alone does not result in trimethoprim resistant E. coli cells. Figure adapted from Pelletier et al. (1998).

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