[Effectiveness of expression of the chloramphenicol acetyltransferase gene controlled by foreign regulatory regions in Escherichia coli cells. I. Construction of vectors for the cloning of transcription regulatory elements].
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New plasmids pML2.1 and pML4 were constructed for cloning the transcription regulatory regions. In the pML2.1 the structural part of chloramphenicol acetyltransferase gene of the pBR325 is under control of the lacUV5-promotor. Because the unique BamH1 cleavage site is in the joint region, one may use it for cloning transcription termination regions and selecting recombinant clones with the AprCms phenotype. As for the pML4, the foreign fragment integration is carried directly before the structural part of cat-gene and it is expressed only if the promotor regions are present. The plasmids were sequenced and their restriction maps were established. Small molecular weight (about 2,0 MDa, AprCmr) or only Apr intact genes and convenient disposition of many unique cleavage sites by restriction endonucleases make these plasmids useful for different genetic engineering experiments.Keywords:
Chloramphenicol acetyltransferase
Multiple cloning site
Transcription
Cloning (programming)
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Minimal plasmids play an essential role in many intermediate steps in molecular biology. For example, they can be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for PCR-based mutagenesis methods. A small backbone also opens up for additional unique restriction enzyme cloning sites. Here we describe the generation of pICOz, a 1185-bp fully functional high-copy cloning plasmid with an extended multiple cloning site. We believe that this is the smallest high-copy cloning vector ever described.
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We report the construction of two broad host range promoter-probe plasmid vectors for rapid analysis of promoters in Gram-negative bacteria. The new vectors, pME4507 and pME4510, carry carbenicillin and gentamycin resistance genes, respectively, and are small sized (4 kb) with a flexible multiple cloning site to facilitate directional cloning of putative promoter elements. The vectors allow rapid plate-based screening for promoter activities, using beta-galactosidase as the reporter enzyme. In the absence of an inserted promoter fragment, they display very low background activity, making them a useful tool for analysis of low expression level promoters.
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Carbenicillin
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New plasmids pML2.1 and pML4 were constructed for cloning the transcription regulatory regions. In the pML2.1 the structural part of chloramphenicol acetyltransferase gene of the pBR325 is under control of the lacUV5-promotor. Because the unique BamH1 cleavage site is in the joint region, one may use it for cloning transcription termination regions and selecting recombinant clones with the AprCms phenotype. As for the pML4, the foreign fragment integration is carried directly before the structural part of cat-gene and it is expressed only if the promotor regions are present. The plasmids were sequenced and their restriction maps were established. Small molecular weight (about 2,0 MDa, AprCmr) or only Apr intact genes and convenient disposition of many unique cleavage sites by restriction endonucleases make these plasmids useful for different genetic engineering experiments.
Chloramphenicol acetyltransferase
Multiple cloning site
Transcription
Cloning (programming)
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Plasmid construction by "forced" or "directional" ligation of fragments digested with two different restriction enzymes is highly efficient, except when inhibited digestion of one site favors vector recircularization. Such failures often result because incomplete double digestion is undetected in vector polylinkers or at terminal cloning sites on a PCR fragment. To test cleavage efficiency indirectly, a "monitor" plasmid is added to the digest. In a suitable monitor, the two test sites are separated by enough DNA (approximately 20% of full length) to distinguish the double digest from the failed single digest. To make this applicable to combinations of 32 popular cloning enzymes, we constructed a set of 4 monitors (pDM1, pDM2, pDM3, and pDM4). Each contains three polylinkers separated by stuffer segments of approximately 1 kb. The 32 sites are distributed in the polylinkers such that at least one plasmid in the set is diagnostic for each enzyme pair. The set is designed to be extended to up to 81 sites. A linearized version of the monitor allows for the determination of which of the two enzymes has failed in an incomplete double digest and is also useful when the target DNA is close to the size of the pDM backbone. The plasmids also serve as versatile self-monitoring cloning vectors for any site combination.
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Restriction map
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Subcloning
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Cloning of methylotropic and other Gram negative bacteria's genes was performed using vectors derived from IncP4 plasmids. Plasmids, such s RSF1010 are 8.8 kb in length, have a high copy number and broad host range and can be mobilized efficiently by a number of conjugative plasmids. IncP4 plasmids have relatively few restriction enzyme's targets suitable for cloning. In this paper the construction of versatile and special purpose IncP4 vectors available for cloning DNA into broad range of bacterial species are described. The seria of versatile vectors involves the transposon containing plasmid and two-replicon vectors. In genetic construction of special vector for direct cloning of restriction fragments the genetic regulation elements of Tn 1 were used. On the base of IncP4 replicon special vectors for construction of bank genes (cosmids) and the vectors for cloning of regulation sequence were also constructed.
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Multiple cloning site
Chloramphenicol acetyltransferase
Gene cassette
Expression cassette
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Restriction digest
PstI
Restriction map
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Subcloning
Multiple cloning site
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Restriction site
Shuttle vector
T-DNA Binary system
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