Plasmids and Cloning Vectors Quiz

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Questions and Answers

What is the purpose of insertional inactivation in identifying transformed cells?

  • To produce a color change in transformed cells
  • To introduce the desired gene into the coding region of DNA
  • To inactivate the enzyme β-galactosidase (correct)
  • To identify recombinant colonies

What is the chromogenic substrate used in the insertional inactivation method?

  • Lactose
  • Galactose
  • X-gal (correct)
  • IPTG

What is the role of the Agrobacterium tumefaciens in cloning plants?

  • It is responsible for producing chemicals that the pathogen requires
  • It transforms normal plant cells into a tumor
  • It carries the T-DNA region that is inserted into the plant genome (correct)
  • It is used as a vector to carry the desired gene into plant cells

What is the purpose of an antibiotic resistance gene in a cloning vector?

<p>To provide a selectable marker for identifying transformed cells (C)</p> Signup and view all the answers

What is the function of the origin of replication in a cloning vector?

<p>To allow for the replication of the vector within the host cell (D)</p> Signup and view all the answers

What is the purpose of the cloning site in a vector?

<p>To facilitate the insertion of the desired gene of interest (B)</p> Signup and view all the answers

What is the purpose of the T-DNA region carried by Agrobacterium tumefaciens?

<p>To introduce the desired gene into the plant genome (B)</p> Signup and view all the answers

Which of the following is a common selectable marker used in cloning vectors?

<p>Antibiotic resistance gene (C)</p> Signup and view all the answers

What is the purpose of the β-galactosidase enzyme in the insertional inactivation method?

<p>To produce a color change in transformed cells (A)</p> Signup and view all the answers

What is the role of the origin of replication in a cloning vector?

<p>To allow for the replication of the vector within the host cell (A)</p> Signup and view all the answers

Flashcards

Plasmid origin of replication

A specific DNA sequence that allows plasmid replication within a host cell.

Selectable marker

A gene that helps scientists identify cells that have taken up a plasmid.

Cloning vector

A DNA molecule used to clone a particular gene or DNA segment.

Plant cell transformation

Process of introducing a gene into plant cells using a modified Ti plasmid.

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Insertional inactivation

Stopping a gene's function by inserting a gene into it.

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Competent host cells

Bacterial cells treated to take up foreign DNA.

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Ti plasmid

A plasmid used to transfer DNA into plants.

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Recombinant

A cell containing a gene that is not original to the cell.

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Antibiotic resistance gene

A gene that confers resistance to a specific antibiotic.

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Cloning site

A specific region (sequence) on the vector that facilitates the insertion of DNA.

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Study Notes

Plasmids as Cloning Vectors

  • A plasmid consists of an origin of replication, which allows for the replication of DNA fragments in the host cell.
  • Plasmids also contain an antibiotic resistance gene, which acts as a selectable marker to identify transformed cells.

Features of Cloning Vectors

  • A cloning vector requires an origin of replication (ori) for DNA replication and maintenance.
  • A selectable marker is necessary to identify transformed cells, such as antibiotic resistance genes (e.g., ampicillin, chloramphenicol, tetracycline, or kanamycin).
  • A cloning site is required for the insertion of foreign DNA.

Cloning Process

  • The antibiotic resistance gene is replaced with the desired gene at the cloning site, resulting in the loss of antibiotic resistance.
  • Recombinants can be selected from non-recombinants based on the loss of antibiotic resistance.

Plant Cell Transformation

  • The tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens is modified into a cloning vector for plant cells.
  • The desired gene is introduced along with other required genes into the T-DNA, resulting in plant cell transformation.
  • The Ti plasmid contains genes for growth regulators, cytokinin and auxin, and energy sources for opine catabolism.

Animal Cell Transformation

  • Retroviruses have been modified to act as vectors for animal cells.

Competent Host Cells

  • Bacterial cells must be made competent to take up DNA, which can be achieved through treatment with divalent ions (e.g., calcium ions) and heat shock.
  • Competent cells can be used for DNA insertion through microinjection, biolistics, gene gun, or other methods.

Insertional Inactivation

  • Insertional inactivation is a method used to identify transformed cells by inserting a gene into the coding sequence of an enzyme, β-galactosidase.
  • The resulting inactivation of the enzyme prevents the formation of lactose, allowing for the identification of recombinant colonies.

Vectors for Plant Cloning

  • Agrobacterium tumefaciens is used as a vector for plant cells, carrying the T-DNA that results in plant cell transformation.

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