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1.
Summary This study examined the transfer of the plasmid pBGH1, an expression vector for bovine somatotropin (BST), from Escherichia coli K-12 strain W3110G [pBGH1] to indigenous microorganisms present in flasks containing Missouri River water. Strain LBB269 is a nalidixic acid-resistant derivative of W3110G which was used as a plasmid-free control strain in these studies. Water samples were inoculated with strains W3110G [pBGH1] and LBB269; after 21 days of incubation the number of viable colony-forming units (CFU) of W3110G [pBGH1] and LBB269 were reduced from an initial level of about 1×10 7 CFU per ml to less than 1 CFU per 100 ml. At this time indigenous microbes resistant to both ampicillin and tetracycline (the antibiotic resistance markers on pBGH1) were isolated from 100 ml of water from each of the flasks inoculated with either strain W3110G [pBGH1] or LBB269. Plasmid DNA was isolated from these organisms and examined for sequences containing the gene for BST from pBGH1, using a polymerase chain reaction (PCR) assay. As expected, the day 0 sample from the flask inoculated with E. coli K-12 strain W3110G [pBGH1] gave a positive PCR response and the day 0 sample from the flask inoculated with E. coli K-12 strain LBB269 gave a negative PCR response. All of the day 21 samples containing indigenous microbes isolated from flasks that were inoculated with either W3110G [pBGH1] or LBB269 were negative in the PCR assay, indicating that the target sequence from pBGH1 was not present in any of these indigenous microorganisms. The results of this particular assay indicate that pBGH1 or the portion of pBGH1 including the BST structural gene had not been transferred from W3110G [pBGH1] to indigenous microbial inhabitants of the Missouri River water flasks during this study. 相似文献
3.
Production of green fluorescent protein (GFP) as a model foreign protein using different culture scales under co-expression
of Vitreoscilla hemoglobin (VHb) in the industrial Escherichia coli strain W3110 (a K12 derivative), was examined. It was found that the VHb co-expressing W3110, exhibited an exceptional and
sustained production ability during cell cultures using different scales, while the VHb non-expressing strain showed variable
production levels. This high and sustained production ability indicates that the VHb co-expressing E. coli W3110, could be successfully employed for practical large-scale production cultures without the need for serious consideration
of scale-up problems. 相似文献
4.
The tandem repeats of LFB15(W4,10)-HP(4-16) (LH) gene were cloned into vector pET32a(+) for recombinant expression in Escherichia coli. The E. coli C43(DE3) was successfully used as the expression host to avoid the cell death during induction in E. coli BL21(DE3). Fusion LH dimer was expressed as inclusion body at a portion of 35% of total cell protein and could be well purified
by Ni 2+-chelating chromatography. The recombinant LH was released by the cleavage of 50% formic acid, and its yield reached 11.3 mg/l
with purity of 95%. The MIC 50 of 3.6 and 1.9 μM of recombinant LH against E. coli CMCC 44102 and Bacillus subtilis ATCC 6633 were determined, respectively. The results demonstrated that expression of tandem LH gene in E. coli C43(DE3) and formic acid cleavage would provide a potent efficient platform for the production of interested peptides.
Zi-gang Tian and Tian-tang Dong contributed equally to this paper. 相似文献
5.
Escherichia coli strains W3110 and BL21 were engineered for the production of plasmid DNA (pDNA) under aerobic and transitions to microaerobic conditions. The gene coding for recombinase A (recA) was deleted in both strains. In addition, the Vitreoscilla hemoglobin (VHb) gene (vgb) was chromosomally inserted and constitutively expressed in each E. coli recA mutant and wild type. The recA inactivation increased the supercoiled pDNA fraction (SCF) in both strains, while VHb expression improved the pDNA production in W3110, but not in BL21. Therefore, a codon-optimized version of vgb was inserted in strain BL21recA−, which, together with W3110recA−vgb+, was tested in cultures with shifts from aerobic to oxygen-limited regimes. VHb expression lowered the accumulation of fermentative by-products in both strains. VHb-expressing cells displayed higher oxidative activity as indicated by the Redox Sensor Green fluorescence, which was more intense in BL21 than in W3110. Furthermore, VHb expression did not change pDNA production in W3110, but decreased it in BL21. These results are useful for understanding the physiological effects of VHb expression in two industrially relevant E. coli strains, and for the selection of a host for pDNA production. 相似文献
6.
Escherichia coli WC196, which was obtained from the strain W3110 by nitrosoguanidine mutagenesis as an overproducer of lysine, produced approximately twenty times more cadaverine than did W3110, and had a twenty fold higher level of rpoS gene product, σ 38, than in W3110. Both WC196 and W3110 had a stop codon (TAG) in rpoS at position which corresponds to the 33th residue of σ 38 protein. In addition, WC196 but not W3110 had a mutation in the gene encoding Ser-tRNA ( SerU), called, supD. Analysis of the amino acid sequence of a σ 38 preparation from WC196 showed that the 33th residue of σ 38 is a serine residue. The Δ rpoS Δ cadA mutant of E. coli W3110 harboring the plasmid containing rpoS, in which the TAG codon was converted to a TCG codon for serine-33 residue of σ 38, expressed a significant amount of Ldc and accumulated a large amount of σ 38. However, the Δ rpoS Δ cadA mutant of W3110 with the plasmid containing the intact rpoS from W3110 could synthesize neither σ 38 nor Ldc significantly. 相似文献
7.
Saccharomyces sp. SK0704 (further defined as SK0704) isolated from long-term-ripening kimchi was identified by a biochemical method with
an API kit; its physiology was found to be very similar to that of S. cerevisiae ATCC 26603 (further defined as ATCC 26603), except in terms of starch utilization. SK0704 did not excrete extracellular glucoamylase,
but utilized starch as a sole carbon source under only aerobic conditions. Crude enzyme excreted from SK0704 catalyzed the
saccharification of starch to glucose, but ATCC 26603 did not. The PCR product obtained using the chromosomal DNA of SK0704
and the primers designed on the basis of the extracellular glucoamylase-coding gene of S. diastaticus was homologous with the intracellular sporulation-specific glucoamylase of S. cerevisiae. SDS-PAGE pattern of soluble protein extracted from yeast cells grown on glucose was greatly different from that on starch.
From these results, we proposed that the SK0704 may have a specific physiological function for starch catabolism such as membrane
transport system and intracellular sac-charification of starch. 相似文献
8.
The effects of cell wall mutation on the oxygenation of linoleic acid (M.W. 280) by recombinant Escherichia coli expressing the CYP102A2 gene encoding self-sufficient P450 monooxygenase of Bacillus subtilis was investigated. After the CYP102A2 gene was heterologously expressed in E. coli W3110 and its isogenic lipopolysaccharide (LPS) structural mutant strains, their whole-cell biotransformation activities
were compared. The mutants used in this study had previously been designated as MLK53, MLK1067, and MLK986. These strains
carry one or two defined mutations in the secondary acyl fatty acids of the LPS lipid A constituent. The CYP102A2 gene was
overexpressed in both wild type E. coli W3110 and its mutant strains, with the specific activity ranging from 1.7 to 2.1 U/mg protein. Interestingly, the whole-cell
biotransformation activity of those recombinant biocatalysts differed significantly. Indeed, MLK986 possessing the tetraacylated
LPS showed a higher oxygenation activity of linoleic acid than those in wild type or other mutant strains having hexa- or
penta-acylated LPSs. These results suggest that the biotransformation efficiency of E. coli-based biocatalysts, especially for medium- to large-sized lipophilic organic substrates, can be enhanced via engineering their LPS, which is known to function as a formidable barrier for hydrophobic molecules. 相似文献
9.
The gene ( choB
b
), encoding cholesterol oxidase from Brevibacterium sp . CCTCC M201008, was cloned and sequenced by PCR (GenBank accession number: DQ345780). The gene consists of 1653 base pairs
and encodes a protein of 551 amino acids. ChoB
b
exhibited a homology of 98% with cholesterol oxidase gene from Brevibacterium
sterolicum ATCC 21387. The cholesterol oxidase gene, cloned in the vector pET-28a, was over-expressed in Escherichia
coli BL21–CodonPlus (DE3)-RP grown at 23°C in Luria-Bertani medium containing 50 μM riboflavin, the precursor of the FAD coenzyme
of the enzyme. A maximum activity of 3.7 U/mg was obtained from cell free extract of E. coli BL21-CodonPlus (DE3)-RP harboring the pET-28a- choB b. 相似文献
11.
A new chitinase gene, chi92, encoding the largest known chitinase from Streptomyces olivaceoviridisATCC 11238 was sequenced by means of different PCR-methods. The cloned gene was expressed in E. coliand the recombinant protein could be detected by Western-blot analysis. The multiplicity of chitinolytic enzymes of this strain is discussed. 相似文献
12.
Gram-negative bacteria, including Escherichia coli, release outer membrane vesicles (OMVs) that are derived from the bacterial outer membrane. OMVs contribute to bacterial cell–cell communications and host–microbe interactions by delivering components to locations outside the bacterial cell. In order to explore the molecular machinery involved in OMV biogenesis, the role of a major OMV protein was examined in the production of OMVs from E. coli W3110, which is a widely used standard E. coli K-12 strain. In addition to OmpC and OmpA, which are used as marker proteins for OMVs, an analysis of E. coli W3110 OMVs revealed that they also contain abundant levels of FliC, which is also known as flagellin. A membrane-impermeable biotin-labeling reagent did not label FliC in intact OMVs, but labeled FliC in sonically disrupted OMVs, suggesting that FliC is localized in the lumen of OMV. Compared to the parental strain expressing wild-type fliC, an E. coli strain with a fliC-null mutation produced reduced amounts of OMVs based on both protein and phosphate levels. In addition, an E. coli W3110-derived strain with a null-mutation in flgK, which encodes flagellar hook-associated protein that is essential along with FliC for flagella synthesis, also produced fewer OMVs than the parental strain. Taken together, these results indicate that the ability to form flagella, including the synthesis of flagella proteins, affects the production of E. coli W3110 OMVs. 相似文献
13.
Summary The seg-3 mutant Escherichia coli does not support the maintenance of mini-F plasmid at 42° C. We cloned the chromosomal DNA segment of the wild-type strain W3110 that complements the Seg – phenotype of this mutant. Cleavage mapping of this segment showed that it was derived from the 76-min region of the E. coli chromosome map. Complementation tests using plasmids carrying subcloned DNA segments suggested that the seg-3 mutant carried two mutations that additively affected the maintenance of mini-F plasmid; one was in the ugpA gene and the other was presumably in the rpoH gene. We generated a disrupted ugpA null mutant and found that the mini-F plasmid was unstable in this ugpA null mutant even at 30° C. This suggests that the ugpA gene product is required for the stable maintenance of mini-F plasmid. 相似文献
15.
A cultivation strategy combining the advantages of temperature-limited fed-batch and probing feeding control is presented. The technique was evaluated in fed-batch cultivations with E. coli BL21(DE3) producing xylanase in a 3 liter bioreactor. A 20% increase in cell mass was achieved and the usual decrease in specific enzyme activity normally observed during the late production phase was diminished with the new technique. The method was further tested by growing E. coli W3110 in a larger bioreactor (50 l). It is a suitable cultivation technique when the O 2 transfer capacity of the reactor is reached and it is desired to continue to produce the recombinant protein.Revisions requested 13 April 2005; Revisions received 6 May 2005 相似文献
16.
Clostridium acetobutylicum ATCC 824 was metabolically engineered for improved xylose utilization. The gene talA, which encodes transaldolase from Escherichia coli K-12, was cloned and overexpressed in C. acetobutylicum ATCC 824. Compared with C. acetobutylicum ATCC 824 (824-WT), the transformant bearing the E. coli talA gene (824-TAL) showed improved ability on xylose utilization and solvents production using xylose as the sole carbon source. During the fermentation of xylose and glucose mixtures with three xylose/glucose ratios (approximately 1:2, 1:1 and 2:1), the rate of xylose consumption and final solvents titers of 824-TAL were all higher than those of 824-WT, despite glucose repression on xylose uptake still existing. These results suggest that the insufficiency of transaldolase in the pentose phosphate pathway (PPP) of C. acetobutylicum is one of the bottlenecks for xylose metabolism and therefore, overexpressing the gene encoding transaldolase is able to improve xylose utilization and solvent production. 相似文献
17.
Lignobacter strain K17 is able to degrade aromatic compounds and to fix atmospheric nitrogen. It was proved that capacity for nitrogen fixation by Lignobacter was plasmid mediated. Plasmid pUCS100 (17.5 Mdal) carrying nif genes was transferred from Lignobacter to Escherichia coli SK1592 and Salmonella typhimurium. The transposon Tn9 was translocated to pUCS100 to facilitate selection of Nif + bacteria. E. coli SK1592 harboring the new plasmid (pUCS101) reduced acetylene under anaerobic conditions. Plasmids pUCS100 and pUCS101 were not stably maintained in E. coli and S. typhimurium.Abbreviations Mdal
megadalton
- CsCl-EtBr
caesium chloride ethidium bromide
- m.o.i.
multiplicity of infection 相似文献
19.
Penicillin G acylase ( pac) gene was cloned into a stable asd
+ vector (pYA292) and expressed in Escherichia coli. This recombinant strain produced 1000 units penicillin G acylase g –1 cell dry wt, which is 23-fold more than that produced by parental Escherichia coli ATCC11105. This enzyme was purified to 16 units mg –1 protein by a novel two-step process. 相似文献
20.
Summary We have cloned genes encoding RNase H from Escherichia coli rnh mutants, Salmonella typhimurium and Saccharomyces cerevisiae. Selection was accomplished by suppression of the temperature-sensitive growth phenotype of Escherichia coli strains containing the rnh-339::cat and either recB270 (Ts) or recC271 (Ts) mutations. RNases H from E. coli and S. typhimurium contained 155 amino acid residues and differed at only 11 positions. The S. cerevisiae and E. coli RNases H were about 30% homologous. A comparison of the amino acid sequences of several RNases H from cellular and retroviral sources revealed some strongly conserved regions as well as variable regions; the carboxyl-terminus was particularly variable. The overlapping, divergent promoter gene organization found in E. coli was observed to be present in S. typhimurium. 相似文献
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