Gene Delivery Methods in Bacteria
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Questions and Answers

What is a notable characteristic of Bacillus subtilis that aids in its survival?

  • It has a low gene count.
  • It can form spores. (correct)
  • It is incapable of producing secreted proteins.
  • It has a double membrane.

    • Which promoter type is typically used in inducible expression systems?

  • Lac promoter (correct)
  • Constitutive promoter
  • Constitutional promoter
  • Basal promoter

    • What is a disadvantage of using bacterial expression systems?

  • Limited gene expression
  • Energy drain from strong expression (correct)
  • Easy retention of plasmids
  • Weak constitutional expression

    • E. coli differs from Bacillus subtilis by having how many membranes?

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

    What occurs during the growth phase of bacterial culture?

    <p>No transcription of the targeted gene</p> Signup and view all the answers

    What is indicated by the transformation of LR to BR in the context provided?

    <p>Transformation of DNA from one bacterial state to another</p> Signup and view all the answers

    Which components are involved in the conversion process mentioned in the content?

    <p>INT and XIS</p> Signup and view all the answers

    What role do background growth conditions play in the process described?

    <p>They are not significant in the conversion process</p> Signup and view all the answers

    What is the purpose of selecting AmpR colonies in this process?

    <p>To identify successful transformations</p> Signup and view all the answers

    What does the term 'toxin' refer to in the context of the provided information?

    <p>An agent affecting bacterial growth and conversion</p> Signup and view all the answers

    What is a significant advantage of using Gateway cloning?

    <p>It allows for rapid recombination cloning.</p> Signup and view all the answers

    What is required when performing a Gibson assembly?

    <p>PCR primers with overlapping sequences.</p> Signup and view all the answers

    Which of the following describes the role of restriction enzymes in the Gateway cloning process?

    <p>They facilitate vector linearization.</p> Signup and view all the answers

    How many fragments can be joined using the Gibson assembly technique?

    <p>Up to 15 fragments.</p> Signup and view all the answers

    What is the purpose of introducing att sequences into the vector during the cloning process?

    <p>To increase cloning speed.</p> Signup and view all the answers

    Which of the following is NOT a characteristic of recombination cloning?

    <p>Relies solely on PCR amplification.</p> Signup and view all the answers

    Why is spectinomycin selection important in the cloning process described?

    <p>It allows for selection of successful clones.</p> Signup and view all the answers

    What method is used to join DNA fragments in the described cloning techniques?

    <p>Recombination.</p> Signup and view all the answers

    What is the process of transduction in bacteria?

    <p>The transfer of genetic material from one bacterium to another via a virus.</p> Signup and view all the answers

    Which of the following is NOT a step in the generation of a transgenic organism?

    <p>Creation of synthetic proteins without genetic modification.</p> Signup and view all the answers

    What is one of the advantages of using E.coli as a host for protein expression?

    <p>It is easy to grow and has a short doubling time.</p> Signup and view all the answers

    Which of the following is a basic requirement for protein expression?

    <p>Gene construct for protein expression.</p> Signup and view all the answers

    What are the main components of a gene construct for protein expression?

    <p>Promoter, structural gene, and terminator.</p> Signup and view all the answers

    Which type of cells is traditionally used to produce recombinant proteins?

    <p>Mammalian, yeast, and bacterial cells.</p> Signup and view all the answers

    What was the first licensed human drug produced using recombinant DNA technology in E.coli?

    <p>Insulin (Humulin).</p> Signup and view all the answers

    Which expression system is NOT commonly used for producing recombinant proteins?

    <p>Bovine cells.</p> Signup and view all the answers

    What is a key advantage of using yeast as an expression system?

    <p>Capable of post-translational modifications</p> Signup and view all the answers

    What is a common disadvantage when expressing proteins in yeast?

    <p>Low levels of expressed recombinant proteins</p> Signup and view all the answers

    In the baculovirus-insect cell expression system, what is the significance of the polyhedrin promoter?

    <p>It produces a high level of mRNA in infected cells</p> Signup and view all the answers

    Why is GAPD utilized in yeast expression systems?

    <p>As an essential gene for leucine biosynthesis</p> Signup and view all the answers

    How can yeast be stored for long-term use?

    <p>At -70°C for prolonged periods</p> Signup and view all the answers

    What is a major challenge associated with the purification of proteins expressed in yeast?

    <p>The thick yeast cell wall complicates extraction</p> Signup and view all the answers

    Which statement reflects a misconception about recombinant products from yeast?

    <p>They can perform complex cellular functions</p> Signup and view all the answers

    What is a consideration when using baculovirus for protein expression?

    <p>It does not integrate into the host DNA</p> Signup and view all the answers

    Study Notes

    Gene Delivery in Bacteria

    • Transduction is a natural process where viruses transfer genetic material between bacteria.
    • Methods to introduce recombinant DNA into host cells include: isolating genes, fusing with vectors, delivering recombinant DNA, selecting modified hosts, and breeding for traits.

    Protein Expression Systems

    • Popular systems for recombinant protein production include bacteria, yeast, insect cells, and mammalian cells.
    • Genetic engineering may alter biopathways in these expression systems.

    Requirements for Protein Expression

    • Essential components for protein expression include a gene construct (promoter, structural gene, terminator) and appropriate host cells like E. coli, Bacillus subtilis, yeast, insect cells, and mammalian cells.

    Escherichia coli (E. coli)

    • Fast-growing bacterium with a doubling time of 20 minutes.
    • Contains around 4400 genes and can be preserved at -70°C.
    • First licensed human drug from recombinant DNA, Humulin (insulin), was produced by E. coli in 1982.

    Gene Expression Constructs

    • Constructs for protein expression must contain elements that regulate transcription and terminate mRNA production.
    • A strong promoter can lead to constitutive expression, which may strain the host.

    Inducible Expression System

    • The lac promoter system, when activated by IPTG, allows control over gene transcription at different growth phases.

    Yeast as an Expression System

    • Yeast like Saccharomyces cerevisiae is capable of stable plasmid maintenance and post-translational modifications.
    • Uses essential genes for survival under selective conditions, like GAPD for leucine biosynthesis.

    Advantages of Yeast Expression

    • Established technology for large-scale fermentation with a proven safety record (GRAS status).
    • Capable of post-translational modifications enhancing protein functionality.

    Disadvantages of Yeast Expression

    • Typically results in low levels of expressed proteins.
    • Proteins may be retained in the periplasmic space, complicating purification processes.

    Baculovirus-Insect Cell Expression System

    • Utilizes baculovirus, specific to insects, to introduce genes into host cells.
    • Uses strong Polyhedrin promoter to achieve high levels of mRNA expression, minimizing threats to humans.

    Cloning Methods

    • Techniques like Gibson assembly allow for the rapid joining of multiple DNA fragments for cloning purposes.
    • PCR primers with overlapping sequences are essential for efficient assembly and cloning into vectors.

    Bacillus subtilis

    • Gram-positive bacterium with around 4100 genes, notable for spore-forming capabilities that enhance survival.
    • Often utilized as an industrial host for secreted protein production due to its easier large-scale culturing processes.

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    Description

    This quiz explores various methods of gene delivery in bacteria, focusing on transduction as a means for transferring genetic material. It also discusses the general process of creating transgenic organisms, including gene isolation and fusion techniques. Test your knowledge on these fundamental genetic engineering concepts.

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