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Questions and Answers
What characterizes low copy number plasmids like pSC101?
What is true about ColicinE1 plasmids?
What modification is made to ColE1 wild plasmids for artificial improvement?
Which of the following statements about the PriA-dependent pathway in theta type B plasmids is true?
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In the context of RNA regulation of theta type B plasmids, what is the role of RNAI molecules?
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Which plasmids are stated to have 700 copies due to their ori modifications?
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What is the main consequence of the mutations in pUC19 compared to pMB1?
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How many maximum copies of ColE1 plasmids are reported to be present originally per cell in wild type?
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What is the primary reason Vibrio natriegens is suggested as an alternative to E.coli?
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Which of the following statements accurately describes the initiation of DNA replication?
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What is the process of transferring DNA into cells using a viral agent called?
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Which statement about the plasmid pUC19 is true?
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How do gene-edited crops compare to those produced by recombinant DNA technology regarding WTC and WTP values?
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What is NOT a basic element of plasmids?
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What role does a promoter play in a plasmid?
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Which component is essential for ensuring the successful cloning of DNA in plasmids?
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What role does the origin of replication (Ori) play in a vector's function within a host cell?
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Which type of plasmid exhibits a low copy number and is synchronized with host chromosomal replication?
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What benefit does high A-T and low G-C content provide in the context of Ori?
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What characterizes high copy number plasmids like pUC19?
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What is characteristic of theta plasmids in Enterobacteria?
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Which mechanism is primarily used by type A theta plasmids for replication?
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What is the effect of tight plasmid copy number regulation on gene expression?
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What happens during discontinuous replication on the lagging strand in theta plasmids?
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What characterizes a relaxed plasmid compared to a stringent plasmid?
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Which of the following is a requirement for the replication of stringent plasmids?
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What happens if RNAseH and Pol I are absent during plasmid replication?
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In which scenario would gene propagation or expression most likely occur?
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Which of the following traits is NOT true about the assembly of relaxed plasmids?
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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
-
Low Copy Number: (ex: pSC101)
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.
