cloning vector

Examples of cloning vector in the following topics:

• Hosts for Cloning Vectors

  • The majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) as the host.
  • A very large number of host organisms and molecular cloning vectors are in use, but the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) and a plasmid cloning vector.
  • If the DNA to be cloned is exceptionally large (hundreds of thousands to millions of base pairs), then a bacterial artificial chromosome or yeast artificial chromosome vector is often chosen.
  • Specialized applications may call for specialized host-vector systems.
  • In practice, however, specialized molecular cloning experiments usually begin with cloning into a bacterial plasmid, followed by subcloning into a specialized vector.

• Plasmids as Cloning Vectors

  • Plasmids can be used as cloning vectors, allowing the insertion of exogenous DNA into a bacterial target.
  • All engineered vectors have an origin of replication, a multi-cloning site, and a selectable marker.
  • Modern plasmids generally have many more features, notably a "multiple cloning site"—with nucleotide overhangs for insertion of an insert—and multiple restriction enzyme consensus sites on either side of the insert.
  • Expression vectors require translation of the vector's insert, thus requiring more components than simpler transcription-only vectors.
  • The pGEX-3x plasmid is a popular cloning vector.

• Obtaining DNA

  • Although a very large number of host organisms and molecular cloning vectors are used, the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) and a plasmid cloning vector.
  • The cloning vector is treated with a restriction endonuclease to cleave the DNA at the site where foreign DNA will be inserted.
  • Most modern vectors contain a variety of convenient cleavage sites that are unique within the vector molecule (so that the vector can only be cleaved at a single site) and are not located within the gene of interest to be cloned.
  • Cells harboring the cloning vector will survive when exposed to the antibiotic, while those that have failed to take up cloning vector will die.
  • The former can therefore be amplified and screened for the presence of the gene of interest in the cloning vector by restriction digest analysis.

• Recombinant DNA Technology

  • Although a very large number of host organisms and molecular cloning vectors are in use, the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) and a plasmid cloning vector.
  • The cloning vector is treated with a restriction endonuclease to cleave the DNA at the site where foreign DNA will be inserted.
  • For cloning of genomic DNA, the DNA to be cloned is extracted from the organism of interest.
  • Modern bacterial cloning vectors (e.g. pUC19) use the blue-white screening system to distinguish colonies (clones) of transgenic cells from those that contain the parental vector.
  • The blue-white screen is a screening technique that allows for the detection of successful ligations in vector-based gene cloning.

• Shuttle Vectors and Expression Vectors

  • Therefore, to make the purification process easy, the cloned gene should have a tag.
  • Cloning vectors, which are very similar to expression vectors, involve the same process of introducing a new gene into a plasmid, but the plasmid is then added into bacteria for replication purposes.
  • In general, DNA vectors that are used in many molecular-biology gene-cloning experiments need not result in the expression of a protein.
  • The pGEX-3x plasmid is a popular cloning vector.
  • Please note the presence of a multiple cloning site, a promoter, a repressor, and a selectable marker.

• Bacteriophage Lambda as a Cloning Vector

  • Transduction is the process by which DNA is transferred from one bacterium to another by a virus.It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector.

• Vectors for Genomic Cloning and Sequencing

  • The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes.
  • Simpler vectors called transcription vectors are only capable of being transcribed but not translated: they can be replicated in a target cell but not expressed, unlike expression vectors.
  • Transcription vectors are used to amplify their insert.
  • Modern plasmids generally have many more features, notably including a "multiple cloning site" which includes nucleotide overhangs for insertion of an insert, and multiple restriction enzyme consensus sites to either side of the insert.
  • The vectors can be extracted from the bacteria, and the multiple cloning sites can be cut by restriction enzymes to excise the hundredfold or thousandfold amplified insert.

• Types of Plasmids and Their Biological Significance

  • Plasmids used in genetic engineering are called vectors .
  • The gene is also inserted into a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments.
  • pUC19 is one of a series of plasmid cloning vectors created by Messing and co-workers in the University of California.
  • It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the most widely used vector molecules as the recombinants, or the cells into which foreign DNA has been introduced, can be easily distinguished from the non-recombinants based on color differences of colonies on growth media. pUC18 is similar to pUC19, but the multiple cloning site region is reversed.

• Detecting Uncultured Microorganisms

  • More specifically, the construction of libraries in bacterial artificial chromosomes (BACs) provided better vectors for molecular cloning.
  • Shotgun sequencing and screens of clone libraries reveal genes present in environmental samples.
  • (A) sampling from habitat; (B) filtering particles, typically by size; (C) Lysis and DNA extraction; (D) cloning and library construction; (E) sequencing the clones; (F) sequence assembly into contigs and scaffolds

• Selection

  • Artificial selection is widely used in the field of microbial genetics, especially molecular cloning.
  • Gene cloning and gene/protein tagging is also common.
  • In a conventional molecular cloning experiment, the DNA to be cloned is obtained from an organism of interest.
  • Subsequently, these fragments are then combined with vector DNA to generate recombinant DNA molecules.
  • Cells harboring the vector will survive when exposed to the antibiotic, while those that fail to take up vector sequences die.