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Phage lambda and plasmid expression vectors with multiple cloning sites and lacZ alpha-complementation 总被引:7,自引:0,他引:7
B E Windle 《Gene》1986,45(1):95-99
Two new lambda vectors were constructed which permit cloning of genes that are potentially lethal if cloned in analogous plasmid vectors. lambda DL10 and lambda DL11 contain the alpha-complementing fragment of lacZ and multiple cloning sites found in the polylinker region of M13mp10 and M13mp11, respectively. DNA cloned into the unique cloning sites of these vectors can be detected by inactivation of alpha-complementation. These lambda vectors provide a lac promoter for expression of foreign genes as well as the ability to make fusion proteins. Two plasmid expression vectors, pPR110 and pPR111, were constructed from lambda DL10 and lambda DL11 respectively, and pCQV2. These plasmids, which express lacZ alpha-complementing activity from the lambda PR promoter, contain multiple cloning sites immediately downstream of the PR promoter. They allow cloning of genes under the control of the PR promoter and the plasmid-encoded thermosensitive (cI857) repressor, and allow easy detection of inserted fragments by inactivation of alpha-complementation. 相似文献
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《Gene》1996,179(2):287-289
The pAC92 plasmid is a direct screening cloning vector which allows positive selection of recombinant clones (re-clones). This new high-copy-number plasmid vector encodes ampicillin resistance and carries the Bacillus subtilis α-amylase (α-Amy)-encoding gene (amy) containing a multiple cloning site. The pAC92 plasmid confers to Escherichia coli transformants an amylolytic phenotype easily detected by iodine vapor staining. The re-clones are identified by insertional inactivation of α-Amy activity. During pAC92 construction, a bacterial growth defect was observed in host cells after some modifications of the promoter region that caused the increase in the amy expression. This suicide characteristic permitted the positive selection of re-clones. A second transformation step was performed to enhance the rate of re-clones per plate. 相似文献
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Recombination cloning encompasses a set of technologies that transfer gene sequences between vectors through site-specific recombination. Due in part to the instability of linear DNA in bacteria, both the initial capture and subsequent transfer of gene sequences is often performed using purified recombination enzymes. However, we find linear DNAs flanked by loxP sites recombine efficiently in bacteria expressing Cre recombinase and the lambda Gam protein, suggesting Cre/lox recombination of linear substrates can be performed in vivo. As one approach towards exploiting this capability, we describe a method for constructing large (>1 × 106 recombinants) libraries of gene mutations in a format compatible with recombination cloning. In this method, gene sequences are cloned into recombination entry plasmids and whole-plasmid PCR is used to produce mutagenized plasmid amplicons flanked by loxP. The PCR products are converted back into circular plasmids by transforming Cre/Gam-expressing bacteria, after which the mutant libraries are transferred to expression vectors and screened for phenotypes of interest. We further show that linear DNA fragments flanked by loxP repeats can be efficiently recombined into loxP-containing vectors through this same one-step transformation procedure. Thus, the approach reported here could be adapted as general cloning method. 相似文献
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Strains of Thermus thermophilus produce unique carotenoids called thermozeaxanthins and their colonies are light-yellow pigmented. Here, we developed a new cloning system allowing for the rapid and convenient detection of recombinants by color screening based on carotenoid production in T. thermophilus. We constructed two cloning vectors that overexpress the crtB gene encoding a phytoene synthase under the strong promoter of the slpA gene. Phytoene synthase is one of essential enzymes for the production of carotenoids. We also isolated a carotenoid-overproducing mutant that formed orange colonies. Because disruption of crtB in the carotenoid-overproducing mutant resulted in white colonies, we used the disruptant as a host strain. Whereas transformants carrying a new cloning vector, pTRK1-PRslpA-crtBcas, grew into unusual red-pigmented colonies probably because of the extreme accumulation of thermozeaxanthins, those carrying the vector with a foreign DNA inserts formed white colonies. Thus, recombinants can be detected easily by color screening (red/white screening) in T. thermophilus. This cloning system requires no additional chromogenic substrate in the medium. We also constructed a promoter-probe vector, pTRK1-crtBmcs-PP, employing the open reading frame of crtB with multiple cloning sites. Using this vector, a series of colony-color phenotypes is observed probably depending on promoter activities of foreign DNA inserts, which enables the rapid probing of promoters. These vectors are useful to simplify cloning procedures and to identify the promoters of different strengths in T. thermophilus. 相似文献
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Four nonconjugative broad-host-range cloning vectors were derived from the W-plasmid Sa. They are small (Mr 5.6?7.2 × 106), carry several drug-resistance markers, and allow constructing and screening for recombinant plasmids generated by the restriction enzymes EcoRI, PstI, BglII, HindIII, BamHI and SalI, 相似文献
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Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning 总被引:19,自引:0,他引:19
M J Palazzolo B A Hamilton D L Ding C H Martin D A Mead R C Mierendorf K V Raghavan E M Meyerowitz H D Lipshitz 《Gene》1990,88(1):25-36
We describe the construction and use of two classes of cDNA cloning vectors. The first class comprises the lambda EXLX(+) and lambda EXLX(-) vectors that can be used for the expression in Escherichia coli of proteins encoded by cDNA inserts. This is achieved by the fusion of cDNA open reading frames to the T7 gene 10 promoter and protein-coding sequences. The second class, the lambda SHLX vectors, allows the generation of large amounts of single-stranded DNA or synthetic cRNA that can be used in subtractive hybridization procedures. Both classes of vectors are designed to allow directional cDNA cloning with non-enzymatic protection of internal restriction sites. In addition, they are designed to facilitate conversion from phage lambda to plasmid clones using a genetic method based on the bacteriophage P1 site-specific recombination system; we refer to this as automatic Cre-loxP plasmid subcloning. The phage lambda arms, lambda LOX, used in the construction of these vectors have unique restriction sites positioned between the two loxP sites. Insertion of a specialized plasmid between these sites will convert it into a phage lambda cDNA cloning vector with automatic plasmid subcloning capability. 相似文献
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A versatile ligation-independent cloning method suitable for high-throughput expression screening applications 总被引:5,自引:0,他引:5
Berrow NS Alderton D Sainsbury S Nettleship J Assenberg R Rahman N Stuart DI Owens RJ 《Nucleic acids research》2007,35(6):e45
This article describes the construction of a set of versatile expression vectors based on the In-Fusion™ cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion™ has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His6-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein. The use of a multiple host-enabled vector allows rapid screening in both E. coli and eukaryotic hosts (HEK293T cells and insect cell hosts, e.g. Sf9 cells). These high-throughput screening activities have prompted the development and validation of automated protocols for transfection of mammalian cells and Ni-NTA protein purification. 相似文献
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PBAD-Based Shuttle Vectors for Functional Analysis of Toxic and Highly Regulated Genes in Pseudomonas and Burkholderia spp. and Other Bacteria
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Dongru Qiu F. Heath Damron Takehiko Mima Herbert P. Schweizer Hongwei D. Yu 《Applied microbiology》2008,74(23):7422-7426
We report the construction of a series of Escherichia-Pseudomonas broad-host-range expression vectors utilizing the PBAD promoter and the araC regulator for routine cloning, conditional expression, and analysis of tightly controlled and/or toxic genes in pseudomonads. 相似文献
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ColE1 cloning of a ribosomal RNA promoter region from lambdarifd18 by selection for lambda integration and excision functions. 总被引:7,自引:0,他引:7
The expression of the ribosomal RNA gene carried by the lambda transducing phage lambdarifd18 is shown to be subject to stringent amino acid control. lambdarifd18 DNA was digested with endonuclease EcoRI and ligated to similarly restricted ColE1 plasmid DNA. Selection for expression of lambda integration and excision gene activity carried by the same DNA fragment results in cloning of the promoter proximal portion of the 16S ribosomal RNA gene. The resulting chemera expresses lambda integration and excision functions as well as encoding the promoter proximal half of a 16S ribosomal RNA gene. 相似文献
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Frank J. Gunn† Christopher G. Tate‡ Peter J. F. Henderson 《Molecular microbiology》1994,12(5):799-809
l -Fucose (6-deoxy-l -galactose) is used as sole carbon source by many microorganisms, and its transport into Escherichia coli is mediated by An l -fucose-H+ symport activity, in order to determine the nature of a putative transporter encoded by the E. coli fucP gene and Identify its protein product it was cloned downstream of the inducible T7 RNA polymerase and lambda Ol Pl promoters, induction of the T7 promoter resulted in the expression of [14C]-l -fucose uptake activity and the concomitant expression of a [35S]-Met-labelled 32 kDa protein at levels too tow for detection by staining with Coomassie briiiiant blue or for protein sequencing, induction of the lambda Ol Pl promoter caused the appearance of l -fucose-H+ symport activity and of a Coomassie brilliant blue-stained 32 kDa membrane protein expressed at high levels sufficient for identification as FucP by N-terminal protein sequencing. The FucP protein is, therefore, a sugar-H+ symporter different in amino acid sequence from any other known transporter. These and other results illustrate the general unpredictability of cloning strategies for attempting the amplified expression of membrane transport proteins. 相似文献
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Md. Rezaul Bari Maizom Hassan Naoki Akai Jiro Arima Nobuhiro Mori 《World journal of microbiology & biotechnology》2013,29(4):683-692
The gene encoding 4-N-trimethylaminobutyraldehyde dehydrogenase (TMABaldehyde-DH) from Pseudomonas sp. 13CM, responsible for the conversion of 4-N-trimethylaminobutyraldehyde (TMABaldehyde) to γ-butyrobetaine in the carnitine biosynthesis pathway, isolated by shotgun cloning and expressed in Escherichia coli DH5α. The recombinant TMABaldehyde-DH was purified 19.5 fold to apparent homogeneity by hydrophobic and affinity chromatography and biochemically characterized. The enzyme was found to be a trimer with identical 52 kDa subunits. The isoelectric point was found to be 4.5. Optimum pH and temperature were found respectively as pH 9.5 and 40 °C. The Km values for TMABaldehyde, 4-dimethylaminobutyraldehyde, and NAD+ were respectively, 0.31, 0.62, and 1.16 mM. The molecular and catalytic properties differed from those of TMABaldehyde-DH I, which was discovered initially in Pseudomonas sp. 13CM. The new enzyme, designated TMABaldehyde-DH II, structural gene was inserted into an expression vector pET24b (+) and over-expressed in E. coli BL21 (DE3) under the control of a T7 promoter. The recombinant TMABaldehyde-DH from Pseudomonas sp. 13CM can now be obtained in large quantity necessary for further biochemical characterization and applications. 相似文献
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A phytase gene from Aspergillus niger was isolated and two Escherichia coli expression systems, based on T7 RNA polymerase promoter and tac promoter, were used for its recombinant expression. Co-expression of molecular chaperone, GroES/EL, aided functional cytosolic expression of the phytase in E. coli BL21 (DE3). Untagged and maltose-binding protein-tagged recombinant phytase showed an activity band of ~49 and 92 kDa, respectively, on a zymogram. Heterologously-expressed phytase was fractionated from endogenous E. coli phytase by (NH4)2SO4 precipitation. The enzyme had optimum activity at 50 °C and pH 6.5. 相似文献
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Rafal Krela Elzbieta Poreba Martyna Weglewska Tomasz Skrzypczak Krzysztof Lesniewicz 《Biochimica et Biophysica Acta (BBA)/General Subjects》2019,1863(3):521-527
Background
During standard gene cloning, the recombinant protein appearing in bacteria as the result of expression leakage very often inhibits cell proliferation leading to blocking of the cloning procedure. Although different approaches can reduce transgene basal expression, the recombinant proteins, which even in trace amounts inhibit bacterial growth, can completely prevent the cloning process.Methods
Working to solve the problem of DNase II-like cDNA cloning, we developed a novel cloning approach. The method is based on separate cloning of the 5′ and 3′ fragments of target cDNA into a vector in such a way that the short Multiple Cloning Site insertion remaining between both fragments changes the reading frame and prevents translation of mRNA arising as a result of promoter leakage. Subsequently, to get the vector with full, uninterrupted Open Reading Frame, the Multiple Cloning Site insertion is removed by in vitro restriction/ligation reactions, utilizing the unique restriction site present in native cDNA.Results
Using this designed method, we cloned a coding sequence of AcDNase II that is extremely toxic for bacteria cells. Then, we demonstrated the usefulness of the construct prepared in this way for overexpression of AcDNase II in eukaryotic cells.Conclusions
The designed method allows cloning of toxic protein coding sequences that cannot be cloned by standard methods.General significance
Cloning of cDNAs encoding toxic proteins is still a troublesome problem that hinders the progress of numerous studies. The method described here is a convenient solution to cloning problems that are common in research on toxic proteins. 相似文献20.
Cosmid cloning and transposon mutagenesis in Salmonella typhimurium using phage lambda vehicles 总被引:12,自引:0,他引:12
E. Tapio Palva Peter Liljeström Shigeaki Harayama 《Molecular & general genetics : MGG》1981,181(2):153-157
Summary We have constructed a strain of Salmonella typhimurium which contains the malB region from Escherichia coli and carries the bacteriophage receptor protein in its outer membrane. Phage adsorbs to this strain but cannot grow, thus providing a very useful system for transposon mutagenesis of S. typhimurium using vehicles carrying transposons. This system can also be used for cosmid cloning. 相似文献