Question 1

Plasmid copy-number control: Plasmids maintained at a limited number of copies per cell are referred to as which class?

Accepted Answer

Introduction / Context: Plasmids differ in copy-number control, which affects plasmid yield, metabolic burden on the host, and expression from cloned genes. Understanding the distinction between stringent and relaxed control helps in selecting vectors for expression versus stability. Given Data / Assumptions: Stringent control maintains only a few copies per cell. Relaxed control allows many copies per cell. Copy number influences plasmid yield and host physiology. Concept / Approach: Stringent plasmids (for example, those with pSC101-type origins) replicate in a tightly regulated manner that yields a low copy number. Relaxed plasmids (for example, pMB1/pUC-type) often reach high copy number, especially with mutations that disrupt negative regulation of replication initiation. Step-by-Step Solution: Define stringent vs. relaxed → low-copy vs. high-copy.Map definition to the question → “limited number of copies” equals stringent.Choose the option explicitly naming stringent plasmids. Verification / Alternative check: Plasmid handbooks categorize origins by copy-number behavior; pSC101-derived systems are classic low-copy, while pUC derivatives are classic high-copy. Why Other Options Are Wrong: Relaxed plasmids: opposite behavior (high copy).Cryptic plasmids: phenotype concept, not copy-number control.All of these/mobilizable: do not specifically define low-copy stringent control. Common Pitfalls: Equating antibiotic selection with copy number; copy control is ori-dependent, not marker-dependent. Final Answer: Stringent plasmids (low-copy-number, tightly regulated).

Question 2

Conjugative plasmids in bacterial genetics Which statement best describes the defining genetic feature of conjugative plasmids that enables cell-to-cell DNA transfer?

Accepted Answer

Introduction / Context: Conjugative plasmids are mobile genetic elements that can move directly between bacteria via conjugation. Understanding what makes a plasmid “conjugative” is essential for topics such as horizontal gene transfer, the spread of antibiotic resistance, and molecular cloning strategies. Given Data / Assumptions: Bacterial conjugation requires physical contact, typically via a pilus. Different plasmids may or may not encode the machinery needed for transfer. Some plasmids also carry accessory genes (e.g., antibiotic resistance), but these are not the defining trait. Concept / Approach: Plasmids that are truly conjugative encode a suite of transfer functions, classically referred to as tra genes. These genes direct pilus formation, mating pair stabilization, nicking/transfer of a single DNA strand, and re-circularization in the recipient. Resistance genes are common passengers but are not required for conjugation competence. Step-by-Step Solution: Identify the defining requirement: the genetic ability to self-transfer.Note that this ability is mediated by a group of transfer genes (tra).Conclude that the correct description is “carry transfer genes known collectively as tra genes.” Verification / Alternative check: Classic F plasmid biology demonstrates that tra gene presence correlates with conjugation capability. Nonconjugative plasmids lack these loci and depend on helpers for mobilization. Why Other Options Are Wrong: Antibiotic resistance (option a) is common but not defining. “Do not exhibit resistance” (option b) is unrelated to conjugation status. “Do not carry transfer genes” (option d) describes nonconjugative plasmids. Chromosomal integration (option e) pertains to episomes, not a universal trait. Common Pitfalls: Equating “conjugative” with “resistance plasmid.” Many resistance plasmids are conjugative, but resistance can also be on nonconjugative plasmids or chromosomes. Final Answer: Carry transfer genes known collectively as tra genes

Question 3

Plasmid copy number terminology Plasmids maintained at multiple copies per cell are commonly referred to as which class?

Accepted Answer

Introduction / Context: Plasmids differ in copy number control. This feature influences gene expression level, plasmid stability, and the yield of recombinant products in biotechnology. Correct terminology helps interpret cloning manuals and research papers. Given Data / Assumptions: Some plasmids replicate to many copies (high copy number), others to few (low copy number). Copy number is governed by the plasmid origin and its regulatory circuitry. Terms “stringent” and “relaxed” are historically used to describe this behavior. Concept / Approach: “Relaxed” control permits many replication initiations, leading to high copy numbers (often tens to hundreds per cell). “Stringent” control ties replication more tightly to the cell cycle, yielding low copy numbers (often 1–2 per chromosome equivalent). “Cryptic” refers to plasmids lacking obvious selectable phenotypes, not copy number per se. Step-by-Step Solution: Identify the class associated with multiple copies: high-copy behavior.Map nomenclature: high copy → relaxed; low copy → stringent.Select “Relaxed plasmids.” Verification / Alternative check: Classic examples include pUC (relaxed, high copy) versus F-type plasmids (stringent, low copy). Vendor datasheets confirm this terminology. Why Other Options Are Wrong: Stringent plasmids are typically low copy. Cryptic plasmids lack obvious traits; copy number may vary. Other labels (conditional, mini-F) do not define high copy status. Common Pitfalls: Assuming high copy always yields higher protein; expression can be limited by toxicity or translation resources. Final Answer: Relaxed plasmids

Question 4

Lambda replacement vectors: maximum foreign DNA size For a bacteriophage λ replacement vector, what is the typical maximum size of foreign DNA that can be inserted while still allowing phage packaging?

Accepted Answer

Introduction / Context: Lambda (λ) vectors are classic cloning tools. Two main classes exist: insertion vectors and replacement vectors. Replacement vectors remove a dispensable chunk (“stuffer”) and replace it with foreign DNA. Correct insert sizes are critical for successful packaging into phage heads. Given Data / Assumptions: λ headful packaging tolerates a limited genome length window. Replacement vectors can accept larger inserts than insertion vectors. Typical teaching values place the maximum practical insert for replacement vectors around the low-to-mid 20 kb range. Concept / Approach: λ packaging requires DNA between approximately 78% and 105% of the wild-type length. In replacement vectors, removing the stuffer frees capacity for a larger insert than insertion vectors, commonly enabling 20–25 kb inserts without compromising packaging efficiency. Step-by-Step Solution: Recall λ size constraints and the concept of replacement versus insertion vectors.Identify the typical upper bound taught for replacement vectors: about 20–25 kb.Choose the option matching this practical limit. Verification / Alternative check: Standard molecular cloning texts and vector manuals list ~20–23 kb as a common maximum for λ replacement systems, corroborating this choice. Why Other Options Are Wrong: 18–20 kb is closer to some insertion vector capacities. 25–30 kb and 40–50 kb exceed reliable λ packaging ranges. 8–10 kb is too small for replacement vectors’ potential. Common Pitfalls: Forgetting both lower and upper packaging limits; too small or too large inserts prevent phage assembly. Final Answer: 20–25 kb

Question 5

Plasmid properties: choose the statement that is NOT universally true for all plasmids Which statement below is not universally valid for every plasmid found in nature?

Accepted Answer

Introduction / Context: Plasmids are small genetic elements central to cloning and bacterial evolution. Many textbook descriptions emphasize circular, double-stranded DNA; however, exceptions exist. Recognizing what is universal versus common avoids overgeneralization in exams and lab work. Given Data / Assumptions: Typical plasmids are extrachromosomal and double-stranded DNA. Replication generally uses host polymerases and proteins, though initiation is controlled by the plasmid’s origin. Some plasmids deviate from “closed circular” topology. Concept / Approach: Most plasmids are covalently closed circular (ccc) dsDNA, but linear plasmids (e.g., in Streptomyces and Borrelia) and unusual forms are known. Therefore, stating “always closed and circular” is not universally true. The dependency on host replication machinery is broadly valid, since plasmids rarely encode a full replication apparatus. Step-by-Step Solution: Assess each statement for universality rather than frequency.Identify the common exception: presence of linear plasmids contradicts “always circular.”Conclude that the “always closed and circular” statement is not universally true. Verification / Alternative check: Microbiology literature documents linear plasmids and linear phage-plasmid hybrids; these confirm the exception to circularity. Why Other Options Are Wrong: Extrachromosomal location (a) is definitional for plasmids. Double-stranded DNA (b) is overwhelmingly true; ssDNA “plasmids” are rare and typically viral replicons. Host-dependent replication (c) is broadly true; plasmids encode control but not full replisomes. Having an origin (e) is required for maintenance. Common Pitfalls: Memorizing “circular” as a strict definition; better to treat it as the most common topology, not a rule without exceptions. Final Answer: It is always a closed, circular DNA molecule.

Question 6

Capacity of λ Charon 34 and Charon 35 vectors Up to what insert size can the lambda vectors Charon 34 and Charon 35 reliably clone DNA?

Accepted Answer

Introduction / Context: Charon series vectors are engineered λ phage replacement vectors used historically for genomic libraries. Knowing typical insert capacities is key for choosing appropriate vectors in classical cloning workflows. Given Data / Assumptions: λ replacement vectors accept larger inserts than insertion vectors. Packaging constraints limit total genome length within defined bounds. Charon 34/35 are examples used in many early library constructions. Concept / Approach: For λ replacement vectors like Charon 34 and 35, stuffer removal allows inserts roughly in the 9–20 kb range while maintaining efficient packaging. Inserts smaller than ~9 kb or larger than ~20 kb risk poor packaging and plaque formation failure. Step-by-Step Solution: Match vector class (replacement) to typical capacity.Recall the accepted window for these specific Charon vectors: ~9–20 kb.Select the answer reflecting this range. Verification / Alternative check: Historical protocols and manuals report successful cloning within ~10–20 kb for these vectors, aligning with the chosen range. Why Other Options Are Wrong: 1–2 kb or 6–7 kb are too small (insertion vector territory). 30–35 kb and above exceed λ packaging tolerance. Common Pitfalls: Ignoring both minimum and maximum packaging thresholds leads to failed library construction. Final Answer: 9–20 kb

Question 7

Definition of cosmids Cosmid vectors are best described as which of the following?

Accepted Answer

Introduction / Context: Cosmids bridge properties of plasmids and λ phage by harnessing the λ cos site while retaining plasmid replication. They became popular for constructing genomic libraries with larger inserts than standard plasmids. Given Data / Assumptions: Cos site enables λ headful packaging of DNA into phage particles. Cosmids replicate in cells as plasmids once introduced. They provide selectable markers for maintenance in bacteria. Concept / Approach: By inserting λ cos sequences into a plasmid backbone, cosmids allow in vitro packaging of large recombinant DNA fragments (often 30–45 kb) into λ particles for efficient transduction, followed by plasmid-like replication in E. coli. Step-by-Step Solution: Identify the hybrid nature: plasmid backbone + λ cos site.Recognize function: packaging via λ system, replication as a plasmid in host.Select the option that states this definition accurately. Verification / Alternative check: Classic cosmid vectors (e.g., pJB8) illustrate this architecture and behavior. Why Other Options Are Wrong: Phages lacking cos cannot package via λ system. Lack of selectable markers contradicts common cosmid design. “Cryptic plasmids” or “mini-chromosomes” are unrelated categories. Common Pitfalls: Confusing cosmids with phagemids; phagemids use filamentous phage (f1) ori for ssDNA production, not λ cos. Final Answer: Plasmids that contain a fragment of λ DNA including the cos site

Question 8

Applications of single-stranded DNA vectors Single-stranded vectors are particularly useful for which purposes in molecular biology?

Accepted Answer

Introduction / Context: Vectors that can generate single-stranded DNA (ssDNA), such as M13 derivatives and phagemids with f1 origins, have specialized uses in sequencing, mutagenesis, and probe generation. Understanding why ssDNA is advantageous clarifies vector selection. Given Data / Assumptions: Sequencing methods historically (e.g., Sanger) favored ssDNA templates. Site-directed mutagenesis workflows often require ssDNA intermediates. Probe labeling can be simpler with ssDNA templates. Concept / Approach: ssDNA avoids issues like re-annealing of complementary strands, simplifies primer access, and enables efficient incorporation of modified nucleotides. Phagemids combine plasmid features with filamentous phage origins to produce ssDNA when helper phage is present. Step-by-Step Solution: List common ssDNA vector applications: sequencing, mutagenesis, probes.Confirm that all three are indeed standard uses.Select “All of the above.” Verification / Alternative check: Protocols from classic M13 systems and modern phagemids (e.g., pBluescript with f1 ori) support these applications. Why Other Options Are Wrong: Any single item alone is incomplete; all listed uses apply. “None of the above” contradicts established practice. Common Pitfalls: Confusing ssDNA production (requires helper phage) with routine plasmid prep (dsDNA). Final Answer: All of the above

Question 9

Definition of the “stuffer” fragment in λ replacement vectors In lambda replacement cloning systems, the term “stuffer” refers to which part of the vector DNA?

Accepted Answer

Introduction / Context: Replacement vectors in the λ system swap out a dispensable central region—called the stuffer—for foreign DNA. Recognizing this component is key to understanding how these vectors accommodate larger inserts than insertion vectors. Given Data / Assumptions: λ genome is divided into left and right arms that flank a central stuffer piece. Packaging requires the final recombinant DNA to fall within length limits. The stuffer is designed to be replaced during cloning. Concept / Approach: Because the stuffer is nonessential for phage propagation in the recombinant context, it can be removed to create space for an insert. The two arms plus the insert must satisfy packaging constraints for viable phage production. Step-by-Step Solution: Identify vector layout: left arm + stuffer + right arm.Define stuffer’s role: removable, replaced by insert.Choose the option that states this explicitly. Verification / Alternative check: Maps of replacement vectors clearly label a central “stuffer” region that is excised during library construction. Why Other Options Are Wrong: Left/right arms are retained; they are not the stuffer. MCS or resistance cassettes are separate features. Common Pitfalls: Confusing insertion vectors (which do not remove a stuffer) with replacement vectors. Final Answer: The central fragment that is removed and replaced by the foreign insert

Question 10

What is a phagemid? Choose the most accurate description of a phagemid's genetic architecture and capability.

Accepted Answer

Introduction / Context: Phagemids combine features of plasmids and filamentous phage, allowing researchers to prepare single-stranded DNA for sequencing, mutagenesis, and display technologies while maintaining standard plasmid benefits (selection, high copy number). Given Data / Assumptions: Phagemids retain a bacterial plasmid origin for dsDNA replication. They also include an f1 phage origin that, with helper phage, yields ssDNA. Selectable markers and MCS sites are typical. Concept / Approach: The defining feature is the additional f1 origin. Upon superinfection with a helper phage, the phagemid DNA is packaged as ssDNA and secreted, which can be isolated easily. This capability distinguishes phagemids from ordinary plasmids and from λ-based cosmids. Step-by-Step Solution: Identify key function: production of ssDNA upon helper phage presence.Map this to architecture: plasmid ori + f1 ori in one vector.Select the option that states both plasmid backbone and f1 origin. Verification / Alternative check: Vectors such as pBluescript and pUC derivatives with an f1 origin are widely used as phagemids, validating the description. Why Other Options Are Wrong: λ att or cos sites are λ features; phagemids are based on filamentous phage f1, not λ. Vectors lacking a plasmid origin would not replicate as plasmids. “Defective phage genome” does not match phagemid function. Common Pitfalls: Confusing cosmids (λ cos-based, large insert) with phagemids (f1 ori-based, ssDNA production). Final Answer: A plasmid cloning vector that also carries the filamentous phage (f1) origin, enabling recovery of single-stranded DNA

Question 11

Insert capacity of P1 cloning vectors Approximately what length of foreign DNA can be accommodated by a P1 cloning vector system?

Accepted Answer

Introduction / Context: P1-based cloning systems were developed to handle larger genomic fragments than λ vectors, facilitating genomic library construction and physical mapping prior to BAC/PAC era dominance. Knowing approximate capacities guides vector choice for large-insert projects. Given Data / Assumptions: P1 vectors package DNA using bacteriophage P1 functions. They accept substantially larger inserts than λ vectors. Different P1 derivatives (e.g., PACs) achieve even higher capacities, but the basic system accommodates around ~100 kbp. Concept / Approach: While λ replacement vectors peak around ~20–25 kb, P1 vectors support much larger inserts on the order of ~70–100 kb. PAC (P1-derived artificial chromosomes) can exceed this, but the question targets the canonical P1 range near 100 kbp. Step-by-Step Solution: Contrast λ capacity with P1 systems to frame scale.Recall typical P1 vector insert sizes around ~100 kbp.Select the option closest to this canonical capacity. Verification / Alternative check: Standard references list P1 vectors in the ~70–100 kbp class; options provided include 100 kbp, which is consistent as an approximate capacity. Why Other Options Are Wrong: 50 kbp is too low for the classic upper range of P1 systems. 20 or 10 kbp align more with plasmids/λ insertion vectors. 5 kbp is a small-insert plasmid domain. Common Pitfalls: Confusing P1 with λ or with much larger BACs (which can exceed 150 kbp); ensure the vector class matches the intended insert size. Final Answer: 100 kbp

Cloning Vectors Questions