Recombinant DNA transfer to Escherichia coli of human faecal origin in vitro and in digestive tract of gnotobiotic mice

1994 ◽  
Vol 15 (1-2) ◽  
pp. 79-87 ◽  
Author(s):  
Yvonne Duval-Iflah ◽  
Isabelle Gainche ◽  
Marie-France Ouriet ◽  
Marie-Claire Lett
Keyword(s):  
Escherichia Coli ◽  
Digestive Tract ◽  
Recombinant Dna ◽  
Dna Transfer ◽  
Gnotobiotic Mice

R-plasmic transfer from Serratia liquefaciens to Escherichia coli in vitro and in vivo in the digestive tract of gnotobiotic mice associated with human fecal flora

1980 ◽  
Vol 28 (3) ◽  
pp. 981-990 ◽  
Author(s):  
Y Duval-Iflah ◽  
P Raibaud ◽  
C Tancrede ◽  
M Rousseau
Keyword(s):  
Escherichia Coli ◽  
Digestive Tract ◽  
Antibiotic Administration ◽  
Fecal Flora ◽  
E Coli ◽  
Serratia Liquefaciens ◽  
Fresh Feces ◽  
Gnotobiotic Mice

It was shown that a strain of Serratia liquefaciens harbors a conjugative R-plasmid responsible for reistance to the following 14 antibiotics: ampicillin, carbenicillin, cephalothin, butirosin, neomycin, paramomycin, kanamycin, lividomycin, gentamicin, tobramycin, streptomycin, tetracycline, sulfonamide, and chloramphenicol, which belong to five families, the beta-lactamines, the aminoglycosides, the tetracyclines, the sulfonamides, and the phenicols. Resistance to th 14 antibiotics was cotransferred by in vitro conjugation between S. liquefaciens and strains of Escherichia coli. Mating between S. liquefaciens and E. coli also occurred in vivo, in the digestive tract of axenic mice and gnotobiotic mice associated with the whole human fecal flora. It was also shown that mating between these two strains occurred even when the donor S. liquefaciens strain was only transient in the digestive tract of the gnotobiotic host animals. A dense population of Bacteroides (10(10) viable cells per g of fresh feces) did not hinder this mating. All the matings occurred in the absence of an antibiotic selection pressure, and the resulting transferred strain of E. coli did not have the same colonizing capacity as the recipient parental strain. However, during antibiotic administration to mice, and even after the end of the drug intake, the transconjugant became established in the dominant population and replaced the parental recipient strain.

scholarly journals Escherichia coli Extract-Based Cell-Free Expression System as an Alternative for Difficult-to-Obtain Protein Biosynthesis

2020 ◽  
Vol 21 (3) ◽  
pp. 928 ◽  
Author(s):  
Sviatlana Smolskaya ◽  
Yulia A. Logashina ◽  
Yaroslav A. Andreev
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Dna ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
Reaction Medium ◽  
Cellular Metabolism ◽  
Heterologous Protein Expression

Before utilization in biomedical diagnosis, therapeutic treatment, and biotechnology, the diverse variety of peptides and proteins must be preliminarily purified and thoroughly characterized. The recombinant DNA technology and heterologous protein expression have helped simplify the isolation of targeted polypeptides at high purity and their structure-function examinations. Recombinant protein expression in Escherichia coli, the most-established heterologous host organism, has been widely used to produce proteins of commercial and fundamental research interests. Nonetheless, many peptides/proteins are still difficult to express due to their ability to slow down cell growth or disrupt cellular metabolism. Besides, special modifications are often required for proper folding and activity of targeted proteins. The cell-free (CF) or in vitro recombinant protein synthesis system enables the production of such difficult-to-obtain molecules since it is possible to adjust reaction medium and there is no need to support cellular metabolism and viability. Here, we describe E. coli-based CF systems, the optimization steps done toward the development of highly productive and cost-effective CF methodology, and the modification of an in vitro approach required for difficult-to-obtain protein production.

Expression of mature pulmonary surfactant-associated protein B (SP-B) in Escherichia coli using truncated human SP-B cDNAs

1990 ◽  
Vol 68 (2) ◽  
pp. 559-566 ◽  
Author(s):  
Li-Juan Yao ◽  
Carol Richardson ◽  
Carol Ford ◽  
N. Mathialagan ◽  
George Mackie ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Blot Analysis ◽  
Pulmonary Surfactant ◽  
Recombinant Dna ◽  
Stop Codon ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
In Vitro Translation ◽  
Protein B

The present communication documents attempts to produce the mature form of human surfactant-associated protein B (SP-B) by modification of the 5′ and 3′ regions of the cDNA and expression of the truncated cDNAs after insertion into the vector pKK223-3. The 5′ end of a cDNA for human SP-B (1407 base pairs) was reconstructed through the ligation of synthetic oligonucleotides to an internal PstI site in the 5′ region. This construction coded for the initiation of protein synthesis at a Met codon adjacent to a codon for the N-terminal Phe of the mature polypeptide. Variable amounts of the 3′ end of the human SP-B cDNA were deleted with mung bean nuclease and exonuclease III. The resulting blunt-ended 3′ fragments were then ligated to a synthetic oligonucleotide linker designed to create a stop codon. The modified 5′ and 3′ ends were ligated to a short PstI-BamHI fragment isolated from the SP-B cDNA and inserted into the expression vector pKK223-3. In vitro translation of sense mRNAs derived from the truncated SP-B cDNAs yielded oligopeptides of appropriate molecular weights, as indicated by urea – sodium dodecyl sulphate –polyacrylamide gel electrophoresis of either intact or immunoprecipitated reaction mixtures. Expression of SP-B in Escherichia coli was confirmed by Northern blot analysis for the mRNAs corresponding to the truncated cDNAs in appropriately transformed bacteria induced with the galactose analog isopropyl-β-thiogalactoside. Western blot analysis using rabbit antisera prepared against bovine SP-B confirmed the presence of mature SP-B in lipid extracts of transformed E. coli, but the amounts were very small. These studies demonstrate the feasibility of producing mature human SP-B through recombinant DNA technology, but indicate that it may be necessary to use a fusion protein approach to obtain sufficient amounts for the formation of artificial pulmonary surfactant.Key words: pulmonary surfactant (human), hydrophobic proteins, artificial pulmonary surfactant, in vitro translation.

scholarly journals Requirement of Purine and Pyrimidine Synthesis for Colonization of the Mouse Intestine by Escherichia coli

2010 ◽  
Vol 76 (15) ◽  
pp. 5181-5187 ◽  
Author(s):  
Jacqueline Vogel-Scheel ◽  
Carl Alpert ◽  
Wolfram Engst ◽  
Gunnar Loh ◽  
Michael Blaut
Keyword(s):  
Escherichia Coli ◽  
Differentially Expressed ◽  
Wild Type ◽  
Gene Encoding ◽  
E Coli ◽  
Pyrimidine Synthesis ◽  
Key Enzyme ◽  
Mouse Intestine ◽  
Gnotobiotic Mice

ABSTRACT To study the adaptation of an intestinal bacterium to its natural environment, germfree mice were associated with commensal Escherichia coli MG1655. Two-dimensional gel electrophoresis was used to identify proteins differentially expressed in E. coli MG1655 collected from either cecal contents or anaerobic in vitro cultures. Fourteen differentially expressed proteins (>3-fold; P < 0.05) were identified, nine of which were upregulated in cecal versus in vitro-grown E. coli. Four of these proteins were investigated further for their role in gut colonization. After deletion of the corresponding genes, the resulting E. coli mutants were tested for their ability to colonize the intestines of gnotobiotic mice in competition with the wild-type strain. A mutant devoid of ydjG, which encodes a putative NADH-dependent methylglyoxal reductase, reached a 1.2-log-lower cecal concentration than the wild type. Deletion of the nanA gene encoding N-acetylneuraminate lyase affected the colonization and persistence of E. coli in the intestines of the gnotobiotic mice only slightly. A mutant devoid of 5′-phosphoribosyl 4-(N-succinocarboxamide)-5-aminoimidazole synthase, a key enzyme of purine synthesis, displayed intestinal cell counts >4 logs lower than those of the wild type. Deletion of the gene encoding aspartate carbamoyltransferase, a key enzyme of pyrimidine synthesis, even resulted in the washout of the corresponding mutant from the mouse intestinal tract. These findings indicate that E. coli needs to synthesize purines and pyrimidines to successfully colonize the mouse intestine.

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