共查询到20条相似文献,搜索用时 62 毫秒
1.
Daniel Cazorla Neus Ferrer-Miralles Jordi X. Feliu Xavier Carbonell Antonio Villaverde 《Biotechnology letters》2001,23(10):831-838
During growth of recombinant bacteria, irregular plasmid partitioning generates non-productive, plasmid-free cells whose proportion usually increases in the culture. For Escherichia coli producing engineered -galactosidases, we have shown a coincidence between plasmid stability and the extension of white/blue areas within individual colonies on X-gal plates. In this context, a good correlation between plasmid permanence in colonies and parameters accurately describing the dynamics of plasmid-free cell population in liquid cultures has been observed. Moreover, the impact of lacZ gene engineering and the metabolic burden imposed by the encoded proteins has been evaluated through plasmid stability by simple image analysis, revealing an enhanced plasmid loss rate as the cells enter into the stationary phase that is modulated by the expression of particular recombinant genes. 相似文献
2.
Summary Inclusion of mycological peptone in the base medium of X-gal plates facilitates the detection of isolates of Saccharomyces cerevisiae producing -galactosidase due to improved colour development. Substitution of tryptone with mycological peptone in a standard Escherichia coli medium (LB) also facilitates colour development on X-gal plates and obviates the need for an inducer. The addition of mycological peptone to the base medium of X-gal plates offers a cost-effective method to improve the detection of the -galactosidase gene in recombinant strains of S. cerevisiae and E.coli. 相似文献
3.
Summary A -galactosidase gene from Lactobacillus sake coding for lactose hydrolysis was cloned and expressed in Escherichia coli. Chromosomal DNA from L. sake was partially digested with the restriction enzyme Sau3AI, and the 3–6 Kb fragment was ligated to the cloning vector pSP72 digested with BamHI. One E. coli transformant expressing -galactosidase was isolated on X-gal plates. It contained a plasmid with an insertion of approx. 4 Kb. The restriction map of the recombinant plasmid was constructed. The characteristics of the recombinant -galactosidase were compared with those of the wild type. The optima pH and temperature for both enzymes was 6.5 and 50°C, respectively. Stability of the enzymes at different temperatures and activity on lactose were determined. 相似文献
4.
G. R. Zealey A. R. Goodey J. R. Piggott M. E. Watson R. C. Cafferkey S. M. Doel B. L. A. Carter A. E. Wheals 《Molecular & general genetics : MGG》1988,211(1):155-159
Summary A 2 m circle-based chimaeric plasmid containing the yeast LEU2 and the Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) genes was constructed. Transformants grown under selective conditions for the LEU2 gene harboured the plasmid at about 15 copies per cell whilst selection for the HSV-1 TK gene led to an increase to about 100 copies per cell. Furthermore, the plasmid copy number could be controlled by the stringency of selection for the TK gene, and the increase in TK gene dosage was reflected in an increase in intracellular thymidine kinase activity. The mitotic stability of the plasmid in high-copy and low-copy number cells was determined. High-copy number cells showed a greater mitotic stability. The relationship of TK expression to plasmid copy number may be useful for the isolation of plasmid copy number mutants in yeast and the control of heterologous gene expression. 相似文献
5.
Improved method for constructing plant amiRNA vectors with blue-white screening and MAGIC 总被引:1,自引:0,他引:1
Artificial microRNA (amiRNA) technology is a novel tool in reverse genetic research for discovering or validating gene functions
in plants. A convenient cloning strategy has been developed to construct plant amiRNA vectors based on lacO reconstruction and mating-assisted, genetically-integrated cloning (MAGIC). The amiRNA precursor fragment was generated by
PCR and inserted into a small donor plasmid through reconstruction of integrated lacO sequence. Blue recombinants were selected on plates containing X-gal and the efficiency of successful clones was 100%. The
amiRNA expression cassette was transferred from the donor plasmid to the recipient plasmid p1301-gfp through MAGIC and an
amiRNA expression plasmid was created. More than 40 plant amiRNA vectors were generated through this method, one of which
was transformed into Arabidopsis thaliana and the target gene was silenced efficiently. The approach will be useful for amiRNA expression vectors construction in plants. 相似文献
6.
Genetic approaches for studying rhizosphere colonization 总被引:3,自引:0,他引:3
Most bacterial traits involved in colonization of plant roots are yet to be defined. Studies were initiated to identify genes in Pseudomonas which play significant roles in this process. The general approach is to use transposons to construct collections of insertion mutants, each of which is then screened for alterations in its interactions with the host plant. In one study a Tn5 derivative containing a constitutively expressed -galactosidase (lacZ) gene was used to generate a collection of insertion mutants which could be distinguished from the wild-type parent on X-gal plates. Each mutant was examined for its ability to colonize wheat seedlings in the presence of the wild-type parent. Mutants which gave wild-type:mutant ratio of 20:1 or greater were obtained. In a second study a Tn5 derivative which carries a promoterless lacZ gene located near one end of the transposon was constructed. Expression of the lacZ gene depends on the presence of an active promoter outside of the transposon in the correct orientation. Insertion mutants generated with this transposon were examined for changes in -galactosidase expression in the presence and absence of plant root exudate. A number of mutants which showed differential lacZ expression have been identified. 相似文献
7.
Shivendra V. Sahi Ronald W. Gagliardo Mary-Dell Chilton William Scott Chilton 《Plant cell reports》1994,13(9):489-492
Summary A method is described for detecting plant metabolites capable of inducing the virulence genes of Agrobacterium tumefaciens. The method uses A. tumefaciens containing a plasmid with an inducible virulence gene fused to a galactosidase gene (virE::lacZ). Thin layer chromatography plates are overlayed with agar containing the indicator bacterium and a chromogenic galactoside (X-gal). Virulence gene inducing plant metabolites induce galactosidase which releases an aglycone readily oxidized by air to a blue pigmented zone at the Rf of the inducer. The method has been used to demonstrate the presence of virulence gene inducers in corn, wheat and rye. The uninduced background level of galactosidase also permits detection of bacterial growth inhibitors after a longer incubation period.Abbreviations X-gal
5-Bromo-4-chloro-3-indoxyl ß-D-galactopyranoside
- TLC
Thin Layer Chromatography
- AS
Acetosyringone
- DIMBOA
2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one
- MBOA
6-Methoxy-2(3H)-benzoxazolone
- T-DNA
Transfer DNA
- Ti Plasmid
Tumor-inducing Plasmid 相似文献
8.
A dehalogenase gene specifying the utilization of a variety of haloacids byPseudomonas sp. Strain 19S has been cloned and expressed inE. coli. Our cloning strategy employed specific amplification of a fragment homologous toPseudomonas dehalogenase gene by Polymerase Chain Reaction (PCR). The PCR amplicon successfully acted as a probe to detect the dehalogenase gene in the Southern Blot of the digestedPseudomonas total DNA. Corresponding fragments were cloned into pUC 18 vector and amplified inE. coli MV 1190. One clone with a substantial dehalogenation activity carried a recombinant plasmid containing a 5.5 kb insert.Abbreviations 2-CPA
2-chloropropionate
- MCA
monochloro acetate
- IPTG
isopropyl-1-thio--D-galactoside
- NBT
nitroblue tetrazolium salt
- PCR
polymerase chain reaction
- X-gal
5-bromo-4-chloro-3-indolyl--D-galactoside
- X-phosphate
5-bromo-4-chloro-3-indolyl phosphate 相似文献
9.
Edwin J. Mientjes Joost H. M. van Delft Brenda M. op't Hof Jan A. Gossen Jan Vijg Paul H. M. Lohman Robert A. Baan 《Transgenic research》1994,3(1):67-69
The determination of thelacZ mutant frequency in gt10lacZ phage vectors isolated from the transgenic mouse strain 40.6 (MutaMouse), requires the screening of large numbers of phages on -galactosidase activity. Existing methods rely on distinguishing a few white plaques on X-gal containing plates amongst a multide of blue ones which is both time-consuming and expensive. The new screening method described here employs the galactose sensitiveEscherichia coli C lacZ recA galE strain into which a multicopy plasmid has been introduced, which results in over-expression of thegalK andgalT genes. In the presence of phenyl--d-galactopyranoside, a substrate for -galactosidase, this leads to the suppression of lacZ
+ phage propagation without affecting the ability of lacZ
– phages to form plaques. With this method it is possible to screen 1.5×106 phages on a single 9-cm Petri dish. Furthermore, the need for blue/white screening has been eliminated. 相似文献
10.
Atsuko Takagi Elizabeth N. Chua Chuenchit Boonchird Satoshi Harashima Yasuji Oshima 《Applied microbiology and biotechnology》1985,23(2):123-129
Summary The cellular -galactosidase activities produced by thelac'Z gene ofEscherichia coli, cloned on YEp, YRp, or YCp-type plasmids in host cells ofSaccharomyces cerevisiae with different ploidies, and which was expressed by a modified jeastHIS5 promoter, showed characteristic differences depending on the plasmid. But for any given plasmid, the isogenic diploid and tetraploid transformants showed slightly lower enzyme activities than their respective haploid transformants. This was due to the similar copy numbers of the plasmids in host cells. Since the cell number per unit volume of the culture decreased with increasing cell ploidy, the enzyme activity per unit volume of the culture decreased significantly. The holding stability of plasmids increased with increasing ploidy of the host cell, especially that of the YRp plasmid. On the YRp plasmid, thelac'Z gene showed higher productivity withTRP1 thanLEU2 as the selection marker for the plasmid. 相似文献
11.
David Gems Aleksey Aleksenko Leonid Belenky Scott Robertson Martin Ramsden Yuri Vinetski A. John Clutterbuck 《Molecular & general genetics : MGG》1994,242(4):467-471
We describe a new method of gene cloning by complementation of mutant alleles which obviates the need for construction of a gene library in a plasmid vector in vitro and its amplification in Escherichia coli. The method involves simultaneous transformation of mutant strains of the fungus Aspergillus nidulans with (i) fragmented chromosomal DNA from a donor species and (ii) DNA of a plasmid without a selectable marker gene, but with a fungal origin of DNA replication (helper plasmid). Transformant colonies appear as the result of the Joining of chromosomal DNA fragments carrying the wild-type copies of the mutant allele with the helper plasmid. Joining may occur either by ligation (if the helper plasmid is in linear form) or recombination (if it is cccDNA). This event occurs with high efficiency in vivo, and generates an autonomously replicating plasmid cointegrate. Transformants containing Penicillium chrysogenum genomic DNA complementing A. nidulans niaD, nirA and argB mutations have been obtained. While some of these cointegrates were evidently rearranged or consisted only of unaltered replicating plasmid, in other cases plasmids could be recovered into E. coli and were subsequently shown to contain the selected gene. The utility of this instant gene bank technique is demonstrated here by the molecular cloning of the P. canescens trpC gene. 相似文献
12.
Madeleine Coulon-Bublex Nicole Mounier Pierre Couble Jean Claude Prudhomme 《Development genes and evolution》1993,202(2):123-127
Summary Freshly deposited eggs ofBombyx mori were microinjected with supercoiled plasmid DNA which carried the -galactosidase coding sequence ofEscherichia coli inserted in place of the coding sequence of theB. mori cytoplasmic actin A3 gene. Transient expression of this fusion gene in the embryo was determined by in situ histochemical detection of enzyme activity. After injection of the plasmid at different stages of embryonic development, -galactosidase activity depending on the injected DNA was only detected in the vitellophages. This indicates the presence of active transactivators of the actin A3 gene promoter in this cell type. Tissue specificity of the fusion gene expression could be related to the early polyploidization of vitellophages, a process which would favour the stability of the nuclear pool of injected plasmids. The activity of the transgene in vitellophages was detectable at 24–33 h of egg development, the stage presumed for the onset of zygotic gene expression, up to the end of embryogenesis. This gene transfer system is thus promising to analyse thecis regulatory sequences of the actin A3 gene and could be utilized for other ubiquitous genes.
Correspondence to: M. Coulon-Bublex 相似文献
13.
Summary The RAD50 gene was cloned as a 4.8 kb fragment in the 2 derived plasmid pFL1. The gene resides in a 3.9 kb segment that was subcloned into the plasmid YRp7. The cloned gene complements the deficiency caused by the rad50-1 mutation with respect to -rays, MMS resistance and UV-induced mitotic recombination. Restoration of the Rad+ phenotype occurs when the cloned gene is on a freely replicating multiple-copy plasmid or in the integrated form.Mapping of the cloned gene following integration of the 2 plasmid, and of the subclone in plasmid YRp7, showed it to be located on the left arm of chromosome XIV. Tetrad analysis of various crosses involving tow different strains carrying rad50-1 showed the mutation to map next to pet2 on chromosome XIV, and not on the right arm of chromsome IV, as previously published. 相似文献
14.
Summary Allele transfer (conversion) was analyzed in transformations with a CmR determining hybrid plasmid, which contained a chromosomal gene controlling threonine prototrophy. In transformations, where a thr
+-cell was transformed with the thr
- plasmid, the chromosomal allele was efficiently converted to the plasmid genotype. This process of gene conversion was rec dependent and greatly enhanced when monomeric rather than unfractionated plasmid DNA was used. 相似文献
15.
Alexis Harington Terence G. Watson Maureen E. Louw Jill E. Rodel Jennifer A. Thomson 《Applied microbiology and biotechnology》1988,27(5-6):521-527
Summary We have studied the stability during fermentation of a hybrid plasmid carrying a Bacillus -amylase gene in Bacillus subtilis. In the absence of antibiotic selection plasmid loss was associated largely with the post-exponential phases of growth and decline. In fermentations containing selective antibiotics, various deleted plasmids were recovered during late stationary phase, regardless of whether the host was rec
+ or recE. We therefore propose that the plasmid loss observed during late growth in antibiotic-free fermentations is due to deletion events which include the origin of plasmid replication. The structure of the deleted plasmids was determined and the sequences in the vicinity of the end-points analysed. When the deleted plasmids were subjected to further fermentations in the absence of selective antibiotics, they were completely stable. 相似文献
16.
A. M. Cashmore M. S. Albury C. Hadfield P. A. Meacock 《Molecular & general genetics : MGG》1986,203(1):154-162
Summary Stable maintenance of the yeast 2 m circle requires an origin of replication and three additional plasmidencoded functions. These are the two trans-acting products of plasmid genes B and C, and the cis-acting region designated STB or REP3. We have carried out detailed studies of the mitotic stability and copy number of 2 m-based plasmids in [cir+] and [cir0] hosts. The results obtained demonstrate that all three of these functions play a major role in plasmid partitioning. The behaviour of hybrid plasmids carrying various regions of the 2 m circle has also been studied in [cir0] isolates containing chromosomally integrated copies of the B and/or C genes. The results demonstrate that the efficiency of partitioning is dependent upon the dosage of gene B but is independent of the dosage of gene C. 相似文献
17.
Hugues Blanc Claude Gerbaud P. Piotr Slonimski Michel Guérineau 《Molecular & general genetics : MGG》1979,176(3):335-342
Summary By using two chimeric plasmids containing yeast URA3 gene as a selection marker and 2 m yeast DNA linked to the bacterial plasmid pCR1, a yeast strain devoid of any 2 m DNA sequence was transformed. Recovery in E. coli of plasmids from yeast transformants showed that the 2 m-less strain was able to maintain the chimeric plasmids as autonomous replicons, with very infrequent plasmid recombination. Hybridization experiments gave no evidence for integration of the URA3 DNA sequence in the chromosomal DNA. The transformed clones showed a high stability of the ura+ character during vegetative multiplication, even in the absence of selective pressure. The specific activity of orotidine 5 monophosphate decarboxylase (coded by the URA3 gene) was 5 to 10 fold higher than in the wild type.These features should offer new possibilities for cloning with yeast. 相似文献
18.
Carolyn Russell Audrey Jarvis Pak-Lam Yu John Mawson 《Applied microbiology and biotechnology》1993,40(2-3):306-308
The plasmid pCR1 has been constructed to express a wheat -amylase enzyme in Kluyveromyces lactis strains. The contruct is based on the vector pCXJ-kan1, which has been derived from pDK1, a native plasmid of K. lactis var. drosophilarum containing the essential regions for plasmid replication and stability. Contruct pCR1 produces an -amylase by DNA isolated from a wheat cDNA clone and is controlled by a Saccharomyces cerevisia PGK promoter.
Correspondence to: C. Russell 相似文献
19.
Latha Khatri Lalithakumari Damodharan 《World journal of microbiology & biotechnology》2001,17(5):523-528
Transfer of a catabolic plasmid from Pseudomonas putida to indigenous marine bacteria and obligate halophilic bacteria was carried out under both in vitro and in situ conditions. The marine recipients, which could not otherwise grow on hydrocarbon substrates, were able to degrade them after the horizontal transfer of the catabolic plasmid from P. putida. Mating conducted on nutrient plates yielded comparatively more transconjugants than in broth mating under laboratory conditions (106 c.f.u./ml). The transconjugants stably maintained the plasmid when they were maintained in seawater amended with selective pressure (antibiotics/Hg (25 g/l) even after 30 days, whereas under non-selective conditions they progressively lost the plasmid after 24 days. The expression of the plasmid in the marine recipients was investigated by gas chromatographic analysis. The overall objective of this study is to evolve a novel strategy for bioremediation of oil spills and the results of the present study suggest that the present approach would offer a better solution for the removal of harmful substances from the environment by avoiding serious interference with the microbial flora of the ecosystem. 相似文献
20.
The site-specific integration of the phage CTX genome, which carries the gene for a pore-forming cytotoxin, into the Pseudomonas aeruginosa chromosome was analysed. The 1,167 by integrase gene, int, located immediately upstream of the attachment site, attP, was characterized using plasmid constructs, harbouring the integration functions, and serving as an integration probe in both P. aeruginosa and Escherichia coli. The attP plasmids p1000/p400 in the presence of the int plasmid pIBH and attP-int plasmids pINT/pINTS can be stably integrated into the P. aeruginosa chromosome. Successful recombination between the attP plasmid p1000 and the attB plasmid p5.1, in the presence of the int plasmid pIBH in E. coli HB101 showed that the int gene is active in trans in E. coli. The int gene product was detected as a 43 kDa protein in E. coli maxicells harbouring pINT. Proposed integration arm regions downstream of attP are not necessary for the integration process. pINT and phage CTX could be integrated together into P. aeruginosa chromosomal DNA, yielding double integrates. 相似文献