Genetic Engineering and Plasmids Overview
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Questions and Answers

What characterizes low copy number plasmids like pSC101?

  • They replicate independently of the host cell's initiation proteins.
  • They have a high concentration of plasmids in each cell.
  • Their copy number is regulated by the host cell's initiation proteins. (correct)
  • They do not utilize replication initiation proteins.

    • What is true about ColicinE1 plasmids?

  • They typically have a low copy number in each cell.
  • They are classified as stringent plasmids.
  • They can kill other bacteria by causing nutrient leakage. (correct)
  • They are derived from Gram-positive bacteria.

    • What modification is made to ColE1 wild plasmids for artificial improvement?

  • Increased plasmid copy number without changes.
  • Insertion of an antibiotic resistance gene. (correct)
  • Addition of a replication initiation protein.
  • Removal of the origin of replication (ori).

    • Which of the following statements about the PriA-dependent pathway in theta type B plasmids is true?

    <p>It is involved in replication fork arrest.</p> Signup and view all the answers

    In the context of RNA regulation of theta type B plasmids, what is the role of RNAI molecules?

    <p>They negatively regulate the replication process.</p> Signup and view all the answers

    Which plasmids are stated to have 700 copies due to their ori modifications?

    <p>pUC19</p> Signup and view all the answers

    What is the main consequence of the mutations in pUC19 compared to pMB1?

    <p>Increased promoter activity.</p> Signup and view all the answers

    How many maximum copies of ColE1 plasmids are reported to be present originally per cell in wild type?

    <p>40 copies</p> Signup and view all the answers

    What is the primary reason Vibrio natriegens is suggested as an alternative to E.coli?

    <p>It can tolerate high salt concentrations.</p> Signup and view all the answers

    Which of the following statements accurately describes the initiation of DNA replication?

    <p>It begins with protein binding to specific DNA motifs.</p> Signup and view all the answers

    What is the process of transferring DNA into cells using a viral agent called?

    <p>Transduction</p> Signup and view all the answers

    Which statement about the plasmid pUC19 is true?

    <p>It is routinely used in protein expression.</p> Signup and view all the answers

    How do gene-edited crops compare to those produced by recombinant DNA technology regarding WTC and WTP values?

    <p>Gene-edited crops have higher WTC and WTP values.</p> Signup and view all the answers

    What is NOT a basic element of plasmids?

    <p>Ribosomal RNA</p> Signup and view all the answers

    What role does a promoter play in a plasmid?

    <p>It initiates the transcription of DNA to mRNA.</p> Signup and view all the answers

    Which component is essential for ensuring the successful cloning of DNA in plasmids?

    <p>Multiple cloning site</p> Signup and view all the answers

    What role does the origin of replication (Ori) play in a vector's function within a host cell?

    <p>It ensures self-replication using host machinery.</p> Signup and view all the answers

    Which type of plasmid exhibits a low copy number and is synchronized with host chromosomal replication?

    <p>pSC101</p> Signup and view all the answers

    What benefit does high A-T and low G-C content provide in the context of Ori?

    <p>Facilitates easier separation of strands during replication.</p> Signup and view all the answers

    What characterizes high copy number plasmids like pUC19?

    <p>Functions independently from the host chromosomal replication.</p> Signup and view all the answers

    What is characteristic of theta plasmids in Enterobacteria?

    <p>They generate replication bubbles resembling the Greek letter θ.</p> Signup and view all the answers

    Which mechanism is primarily used by type A theta plasmids for replication?

    <p>DnaA dependent pathway.</p> Signup and view all the answers

    What is the effect of tight plasmid copy number regulation on gene expression?

    <p>It directly influences gene dosage affecting both plasmid and host.</p> Signup and view all the answers

    What happens during discontinuous replication on the lagging strand in theta plasmids?

    <p>Short fragments known as Okazaki fragments are produced.</p> Signup and view all the answers

    What characterizes a relaxed plasmid compared to a stringent plasmid?

    <p>It allows for multiple copies during growth.</p> Signup and view all the answers

    Which of the following is a requirement for the replication of stringent plasmids?

    <p>They depend on sufficient amounts of REP101 protein.</p> Signup and view all the answers

    What happens if RNAseH and Pol I are absent during plasmid replication?

    <p>Replication can still occur at a reduced efficiency.</p> Signup and view all the answers

    In which scenario would gene propagation or expression most likely occur?

    <p>With relaxed plasmids that are independent of host replication.</p> Signup and view all the answers

    Which of the following traits is NOT true about the assembly of relaxed plasmids?

    <p>It involves synchronization with chromosomal DNA.</p> Signup and view all the answers

    Study Notes

    The Bacterium Vibrio Natriegens

    • An alternative to E.coli for genetic engineering
    • Tolerates high salt concentrations
    • Exhibits an ultrafast growth rate
    • Can be grown on lower cost media
    • Not pathogenic to humans

    DNA Replication Initiation

    • Depends on proteins that recognize and bind to a specific DNA sequence motif
    • Occurs in several types of DNA, including:
      • Bacterial chromosomal DNA
      • Theta type A plasmids
      • Low copy number plasmids

    Transduction

    • The transfer of DNA into cells through viral agents

    pUC19 Plasmid

    • A high copy number plasmid
    • Frequently used in protein expression

    Gene Edited Crops

    • Exhibit higher WTC (willingness to consume) and WTP (willingness to pay) values than crops produced using recombinant DNA technology

    Basic Elements of Plasmids

    • Origin of Replication (Ori):
      • Enables self-replication in a host cell
      • Controls the number of plasmid copies per cell
      • High A-T and low G-C content
    • Multiple Cloning Site (MCS):
      • A region containing several restriction enzyme recognition sites
      • Allows for the insertion of foreign DNA fragments
    • Selectable Marker:
      • A gene conferring resistance to an antibiotic
      • Facilitates identification of cells containing the plasmid
    • Promoter:
      • A DNA sequence that initiates transcription of a gene
      • Controls gene expression
    • Sequence Elements of Translation:
      • Sequences necessary for protein synthesis
      • Include ribosome binding sites and start/stop codons

    Function of Basic Elements

    • Origin of Replication (Ori):
      • Ensures self-replication in the host cell using its replication machinery
      • Controls copy number per cell
      • Initiates replication with the help of DNA polymerase enzymes
      • Contains a Replicon - A sequence of DNA that includes the Ori and supplementary sequences
    • Types of Ori:
      • Low Copy Number: (ex: pSC101)
        • Has about 20 copies per cell
        • Tightly controlled
        • Replication is synchronized with host cell chromosomal DNA replication
      • High Copy Number: (ex: pUC19)
        • Has about 700 copies per cell
        • Less tightly controlled
        • Replication is independent from host cell chromosomal replication

    Chromosomal DNA Replication in Prokaryotes

    • The most dominant plasmid type in enterobacteria (gram-negative, rod-shaped, flagella containing, and lactose fermenting)
    • Examples of enterobacteria include Escherichia, Shigella, Salmonella, and Yersinia
    • Replication is coordinated; leading and lagging strands are replicated simultaneously
    • Replicated DNA forms bubbles that resemble the Greek letter θ (theta) under the electron microscope
    • Tight plasmid copy number regulation is crucial because of the impact of plasmid gene dosage on gene expression
    • Two types of host-replication mechanisms exist:
      • DnaA Dependent: Theta Type A
      • PriA-Dependent Pathway: Theta Type B, C, and D
        • Theta type B resembles ColE1-Like plasmids
        • Theta type C resembles ColE2-Like plasmids
        • Theta type D resembles ColE3-Like plasmids

    Theta Type A Plasmids

    • Low copy number plasmids
    • Examples: pSC101
    • Replication initiation proteins are used to limit copy number
    • Replication initiation protein:
      • In chromosomal DNA, it’s DnaA
      • In stringent plasmids, it’s both DnaA (from chromosomal DNA) and Rep (encoded by the plasmid)
        • Rep: Replication-encoded initiation protein
      • Handcuffing is used: Two plasmids and Rep bind to the bacterial chromosome and limit copy number.

    Theta Type B Plasmids

    • Examples: ColicinE1 (ColE1)-producing plasmids of E.coli
    • High copy number
    • Originally, max 40 copies per cell in wild type.
    • Associated with Gram-negative bacteria
    • Used to terminate (kill but not lyse-bactericidal) other types of bacteria by:
      • Inserting into the target bacteria’s plasma membrane
      • Leaking nutrients and protons from the target cell

    Artificial Improvement in ColE1 Wild Plasmid

    • Colicin gene is removed
    • The ori is kept
    • Antibiotic resistance gene (like ampR or bla ) is added
    • Theta plasmid contains ColE1-derived origin of replication (ori)

    Theta Type B Plasmids

    • PriA-dependent Pathway: resembles replication restart after replication fork arrest.
    • RNA primer (RNAII) is encoded by Ori and is approximately 600 nucleotides long
    • Anti-sense RNA (RNAI) molecules negatively regulate the replication process.
    • Examples: pMB1 ori (20 copy), pUC19 (700)
    • pUC19 differs from pMB1 by two mutations:
      • Increased promoter activity
      • Primer less sensitive to inhibition by RNA I
      • Stabilize secondary structure for primer formation

    Theta Type B Plasmids (Independent from Host Cell Replication)

    • An R-loop forms, a structure containing DNA and RNA
    • R-loop formation is the only essential step for replication; if DNA polymerase III, RNAseH, and Pol I are missing, initiation is still possible, albeit at reduced efficiency.

    Relaxed vs. Stringent Plasmids

    • Relaxed Plasmids (Type B):
      • High copy number per cell (ex: 700)
      • Used for gene propagation/expression
      • Less restricted to host cell DNA replication machinery
      • Multiple copies exist at all points in growth
      • PriA-dependent assembly
      • Replication is partially independent from host cell replication
      • RNA molecules are synthesized by the plasmid itself
      • Examples: pBluescript II SK
    • Stringent Plasmids (Type A):
      • Low copy number per cell (ex: 20)
      • More likely to work with toxic protein genes
      • More restricted to host cell DNA replication machinery
      • Copies are only made during cell division
      • DnaA-dependent assembly
      • Replication is synchronized with host cell chromosomal DNA replication
      • Requires enough amount of REP101 protein for replication
      • Examples: pSC101

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    Description

    This quiz covers the key concepts related to genetic engineering, focusing on the bacterium Vibrio natriegens, DNA replication initiation, transduction, and plasmid biology. Topics include the unique properties of plasmids like pUC19 and the implications of gene-edited crops. Test your knowledge on these essential molecular biology themes.

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