Molecular Biology Vectors Quiz
42 Questions
3 Views

Choose a study mode

Play Quiz
Study Flashcards
Spaced Repetition
Chat to lesson

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

Which of the following is NOT a basic element of reading vectors?

  • Protein Purification Region (correct)
  • Selectable Marker
  • Multiple Cloning Site (MCS)
  • Origin of replication (Ori)

    • The purpose of cloning vectors is to express proteins.

    False (B)

    What are two components required for the blue/white colony screening method?

    X-Gal and IPTG

    A ______ site allows the insertion of DNA fragments into a vector.

    <p>multiple cloning</p> Signup and view all the answers

    Match the following terms with their descriptions:

    <p>Promoter = Sequence that initiates transcription Selectable Marker = Identifies successful vector incorporation Ori = Allows replication of the vector Recombinant Vector = Vector containing inserted foreign DNA</p> Signup and view all the answers

    What is the function of a selectable marker in a vector?

    <p>To identify cells that contain the vector (D)</p> Signup and view all the answers

    Recombinant vectors can be used for both cloning and expression.

    <p>True (A)</p> Signup and view all the answers

    Identify one essential element that must be present in expression vectors.

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

    What is the primary advantage of tissue-specific promoters in gene therapy?

    <p>They enable targeted overexpression in tumor cells. (D)</p> Signup and view all the answers

    Regular constitutive promoters are effective in directing transgene expression in specific tissue types.

    <p>False (B)</p> Signup and view all the answers

    What is CEA in the context of colorectal cancer?

    <p>Carcinoembryonic Antigen</p> Signup and view all the answers

    Tissue-specific promoters can focus gene expression in the target edible part of the plant, which in this case is the ______.

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

    What is the main function of IPTG in protein expression?

    <p>To induce protein expression by dissociating the repressor from the lac operator (C)</p> Signup and view all the answers

    Cells harboring pHMGWA without the insert survive and produce colonies.

    <p>False (B)</p> Signup and view all the answers

    What is the role of MBP in protein expression?

    <p>To enhance solubility and facilitate purification of target proteins.</p> Signup and view all the answers

    The __________ is a common protein expression tag that enhances solubility.

    <p>Maltose Binding Protein (MBP)</p> Signup and view all the answers

    How is the MBP-fusion protein purified?

    <p>By affinity chromatography using amylose resin (D)</p> Signup and view all the answers

    Only genes between the attR1 and attR2 borders are retained after recombination.

    <p>True (A)</p> Signup and view all the answers

    What is the primary function of the 6xHis tag in recombinant proteins?

    <p>To assist in the purification and detection of the protein (D)</p> Signup and view all the answers

    What happens to the MBP-fusion protein after purification?

    <p>It can be treated with a specific protease to cleave the MBP-tag.</p> Signup and view all the answers

    The His tag can only be attached to the N-terminus of recombinant proteins.

    <p>False (B)</p> Signup and view all the answers

    Name one type of metal ion that a His-tagged protein can bind to for purification.

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

    The Tetracycline-controlled operator system is an example of a _____ inducible gene expression system.

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

    Which of the following configurations of the Tetracycline-controlled operator system is not mentioned?

    <p>Dual-activation configuration (A)</p> Signup and view all the answers

    Anti-His-tag antibodies are available for use in assays involving His-tagged proteins.

    <p>True (A)</p> Signup and view all the answers

    His-tagged proteins can be detected without the use of a _____-specific antibody or probe.

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

    Match the following purification methods to their descriptions:

    <p>Nickel affinity chromatography = Uses nickel ions to bind His-tagged proteins for purification Cobalt affinity chromatography = Utilizes cobalt for purification similar to nickel Copper affinity chromatography = Employs copper ions for binding His tags Ion exchange chromatography = Separates proteins based on their charge</p> Signup and view all the answers

    What is the function of enhancer elements in gene expression?

    <p>Fine-tune gene expression in specific tissues (C)</p> Signup and view all the answers

    Cis-acting elements are important for general gene expression across all tissues.

    <p>False (B)</p> Signup and view all the answers

    What forms a transcriptional initiation complex on the TATA box?

    <p>Transcription factors</p> Signup and view all the answers

    The core promoter is located close to the ____________ initiation sites.

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

    Which of the following describes CpG islands?

    <p>Regions devoid of methylation (B)</p> Signup and view all the answers

    Housekeeping genes require tissue-specific transcription factors.

    <p>False (B)</p> Signup and view all the answers

    Name the sequence that is commonly found in core promoters.

    <p>TATA box</p> Signup and view all the answers

    What mechanism does tetracycline use to eliminate sensitive cells?

    <p>Binds to ribosome and misreads mRNA (C)</p> Signup and view all the answers

    TetR is a repressor that activates gene expression in the presence of tetracycline.

    <p>False (B)</p> Signup and view all the answers

    What is the function of Doxycycline in the tetracycline operon?

    <p>It dissociates TetR from TetO, initiating transcription.</p> Signup and view all the answers

    In a Tet-off configuration, gene expression is ON in the absence of _____ .

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

    Which of the following statements describes the Tet-on configuration?

    <p>Gene expression is active only in the presence of tetracycline. (B)</p> Signup and view all the answers

    The TetO element in the tetracycline operon is where the _____ binds to inhibit gene expression.

    <p>repressor (TetR)</p> Signup and view all the answers

    Match the following terms related to tetracycline configurations with their correct descriptions:

    <p>Tet-off = Expression is ON without tetracycline Tet-on = Expression is ON only when tetracycline is present TetR = Repressor that blocks transcription rtTA = Mutated tTA that requires tetracycline for expression</p> Signup and view all the answers

    What determines tissue-specific expression of a gene?

    <p>The combined activities of cis- and trans-acting elements.</p> Signup and view all the answers

    Study Notes

    Genetic Engineering (GE-1) - Methodology of Gene Manipulation

    • Lecture 7 covered methodology of gene manipulation.
    • The lecture was given by Dr. Mohamed Hazman on November 13, 2024.

    Promoters: Screening and Gene Expression (APB1303, Lecture 7)

    • The lecture discussed promoters and gene expression.
    • Diagrams showed constitutive promoters, teto expression cassette of interest, and the hypertrophic heart (with diseased and normal versions).
    • Various IPTG concentrations (0.05 mM, 0.5 mM, 1 mM) were tested, illustrating how IPTG affects gene expression.
    • The diagrams show RNA polymerase and TetR.

    Reading Vectors: Basic Elements

    • The lecture explained basic elements of reading vectors.
    • Elements included origin of replication (Ori), multiple cloning site (MCS), selectable marker, promoter, and sequence elements of translation.
    • There was a visual representation of a plasmid map, showing various restriction sites (e.g., EcoRI, HindIII, BamHI).
    • The diagram of the plasmid included pBluescript II SK(+).

    Vectors

    • The lecture described vectors for cloning and expression.
    • Vectors are used to create copies of genes, and to express proteins.
    • A diagram of different vectors was provided, and different parts of the vectors were highlighted.
    • Parts of the vector included the origin of replication (ORI), multiple cloning site (MCS), selectable marker, promoter, and elements of translation.

    Cloning and Expression

    • Cloning involves making copies of a gene, and expression involves producing a protein from that gene.
    • Vectors are crucial to achieve both of these tasks in molecular biology.
    • The flow chart linked cloning with the production of proteins, with special elements for protein expression and purification highlighted.
    • The diagrams illustrate the various stages of cloning and expression, including gene of interest, amplification, and protein expression.

    Blue/White Colony for Cloning

    • Blue/white colony screening is used to identify recombinant vectors, and differentiating them from empty vectors.
    • The optimal amounts of IPTG and X-Gal are crucial for efficient blue/white colony screening.

    Rosetta Stone

    • The Rosetta Stone was mentioned but not discussed in detail.

    To Express a Gene

    • A special E. coli strain (like Rosetta) and a specialized expression vector are required to express a gene.
    • The process of using a vector for expression is linked to the presence of chloramphenicol resistance gene within the vector.
    • Production of a toxic protein due to the lack of the insert needs an expression vector.

    IPTG Induces Protein Expression

    • IPTG, isopropyl-β-D-1-thiogalactopyranoside, induces protein expression.
    • IPTG is added in controlled quantities (0.05 mM, 0.5 mM, 1 mM) to illustrate the resulting effect on protein expression.
    • The lac repressor is explained and its role in controlling expression.

    Recombinant Protein Tags

    • Maltose Binding Protein (MBP) is used as a tag for better protein solubility and purification.
    • MBP fusion proteins are purified using amylose resin.
    • 6xHis tag is used for rapid purification, detection and specific quantification.
    • 6xHis tag is useful for use in assays, without using specific antibodies.

    Purification Strategy

    • The lecture presented a diagram illustrating protein purification strategies, including sample loading, washing and elution steps.
    • The procedure clarifies protein purification using MBP (Maltose Binding Protein) affinity tags.
    • There are different stages leading to the purification of the target protein, which is shown in an image format.

    Mammalian Inducible Promoters (Ex2)

    • The expression system uses the Tn10 tetracycline resistance operon.
    • Three configurations were presented: repression-based, Tet-off and Tet-on.
    • The role of IPTG induction of gene expression was included.

    Tetracycline

    • Tetracycline eliminates sensitive cells by binding to ribosomes, mis-reading mRNA and thus disrupting protein synthesis.
    • Tetracycline-resistant E. coli strains/types were presented.
    • Drug efflux mechanism was also mentioned

    Repression-based Configuration

    • A diagram illustrating a repression-based configuration with a constitutive promoter, TetR, Teto, and the gene of interest.
    • The mechanism showed how Dox (Doxycycline) dissociates TetR-teto and initiates RNA transcription.

    Tet-off Configuration

    • Diagram showing Tet-off configuration, with Tet-Response Element (TRE), tTA, and the regulatory mechanisms.
    • The expression is ON in the absence of tetracycline.
    • Adding Tetracycline dissociates the protein and stops the expression.

    Tet-on Configuration

    • Diagram showing Tet-on regulatory mechanisms.
    • The expression is OFF until the presence of tetracycline, which will lead to expression.

    Tissue-Specific Promoters

    • Tissue-specific promoters ensure gene expression is localized to specific tissues or cell types.
    • Cis-acting elements and trans-acting proteins are highlighted and how they interact to control expression.
    • Enhancer elements play an important role in fine-tuning gene expression.
    • The importance of these elements in tissue-specific genetic therapeutic strategies is noted.

    Focused vs Dispersed Transcription

    • The differences between TATA-containing and TATA-less gene promoters were explained with diagrams to illustrate the concepts.
    • Housekeeping and tissue-specific promoters are distinguished.

    CpG Islands and TATA Box

    • CpG islands are regions devoid of methylation, with higher G+C content.
    • They are associated with generic transcription initiation sites (without TATA box).
    • TATA box is associated with basic expression in tissue-specific promoters and less chromatin accessibility.

    Colorectal Cancer-Specific Promoters (Ex1)

    • The prevalence of colorectal cancer was noted, with the benefit of gene suicide and cytotoxic effects highlighted.
    • Therapeutic genes targeting tumor cells, not normal cells, were discussed.

    Specificity and Targeting

    • Diagrams illustrate the mechanisms of targeting therapeutic genes to colorectal cancer cells, avoiding their expression in normal cells.

    Endosperm-Specific Glutelin Promoter

    • The need for tissue-specific promoters, particularly in endsoperm was highlighted.
    • How tissue-specific promoters like glutelin promoters can help to focus transgene expression in the endosperm of seeds, thus minimizing any negative consequences, without unnecessary metabolic penalties, is discussed.

    Thanks and Good Luck

    • A general closing message.

    Studying That Suits You

    Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

    Quiz Team

    Related Documents

    Genetic Engineering (GE1) Lecture 7 PDF

    Description

    Test your knowledge on the basic elements of reading vectors in molecular biology. This quiz covers topics including cloning vectors, selectable markers, and tissue-specific promoters, providing insights into recombinant DNA technology and expression methods. Challenge yourself and see how well you understand these essential concepts.

    More Like This

    Use Quizgecko on...
    Browser
    Open
    Browser
    Browser