Question 1

Cloning tools — classification of the P1 cloning vector in molecular biology  In recombinant DNA technology, the P1 cloning vector belongs to which vector class?

Accepted Answer

Introduction / Context: Large-insert cloning systems enable construction of genomic libraries and manipulation of substantial DNA fragments. P1-based vectors are important historical tools bridging the gap between cosmids and bacterial artificial chromosomes (BACs). Given Data / Assumptions: P1 refers to the bacteriophage P1 system adapted as a cloning vector. It supports relatively large DNA inserts compared with standard plasmids. Classification depends on the biological origin and replication mechanisms of the vector backbone. Concept / Approach: P1 cloning vectors derive from bacteriophage P1 elements. They can package large DNA fragments using phage functions and maintain inserts in Escherichia coli with specialized replication control. Thus, by origin and architecture, P1 vectors are classified within bacteriophage-derived systems rather than simple plasmids, phagemids, or yeast-based artificial chromosomes. Step-by-Step Solution: Identify the name: “P1” points to bacteriophage P1 lineage.Recall function: supports larger inserts than routine plasmids.Classify by origin → bacteriophage-derived vector.Select the matching option “Bacteriophage.” Verification / Alternative check: Molecular cloning manuals and vector catalogs clearly categorize P1 vectors as phage-based, distinct from cosmids (lambda-based hybrids), plasmids, and phagemids (plasmids with filamentous phage origins). Why Other Options Are Wrong: Plasmids (option a) are smaller-capacity circular DNA vectors. Cosmids (option b) are lambda cos-site plasmid hybrids. Phagemids (option d) derive from filamentous phage systems (e.g., M13). YACs (option e) are yeast-chromosome constructs unrelated to P1. Common Pitfalls: Confusing P1 vectors with lambda cosmids; although both are phage-related, P1 is a distinct bacteriophage platform with different packaging and maintenance features. Final Answer: Bacteriophage

Question 2

In molecular cloning, M13 (often written as M13 or M13mp series) is best described as which type of vector and virion morphology? Choose the most accurate characterization for routine lab use.

Accepted Answer

Introduction / Context: M13 is a classic cloning workhorse in molecular biology. Understanding what M13 is—both as a virus and as a vector—helps students connect phage biology with practical applications such as Sanger sequencing, site-directed mutagenesis, and display technologies. This question tests recognition of M13’s morphology and genome type in the context of vector design. Given Data / Assumptions: M13 infects Escherichia coli carrying the F pilus. M13 particles are long and filamentous rather than icosahedral. M13-based vectors (e.g., M13mp, f1-based plasmids) provide single-stranded DNA useful for sequencing and mutagenesis. Concept / Approach: Natural M13 is a filamentous bacteriophage with a circular single-stranded DNA genome. Laboratory derivatives are engineered to facilitate cloning, blue–white screening, and generation of ssDNA. Because M13 produces ssDNA packaged into filamentous particles and secretes virions without lysing the host, it is especially suited to applications needing high-quality ssDNA templates. Step-by-Step Solution: Identify morphology: M13 forms filamentous virions.Identify genome and vector utility: M13 provides ssDNA widely used for sequencing and mutagenesis.Select the option that captures both attributes together. Verification / Alternative check: Standard cloning manuals describe M13 as a filamentous phage producing circular ssDNA and as the basis for M13mp vectors and f1-origin phagemids used to recover ssDNA from cultures after helper phage rescue. Why Other Options Are Wrong: D: A plasmid is a non-viral circular dsDNA replicon; M13 is a phage. E: Cosmids are plasmids with lambda cos sites for packaging large dsDNA fragments, not filamentous ssDNA phage. A or B alone are incomplete; the best answer must cover both properties. Common Pitfalls: Confusing M13 with lambda (a dsDNA head–tail phage) or assuming all phage vectors yield dsDNA only. Final Answer: Both a filamentous phage and an ssDNA vector

Question 3

In plasmid biology, what does the term “plasmid incompatibility” specifically mean under no selection pressure within the same bacterial host cell?

Accepted Answer

Introduction / Context: Plasmid incompatibility groups are central to vector selection in cloning and synthetic biology. If two plasmids share the same replication control system, they compete and one is typically lost unless selection is applied. Understanding incompatibility prevents failed co-transformation experiments and unstable constructs. Given Data / Assumptions: No antibiotic or auxotrophic selection is applied. Two plasmids with similar replication/partition systems are introduced. Host is otherwise permissive for plasmid replication. Concept / Approach: Incompatibility arises when plasmids share control elements (ori and copy control/partitioning). The cell cannot maintain both at stable copy numbers; random segregation and replication control favor dominance of one plasmid lineage. Distinct incompatibility (Inc) groups enable stable coexistence (e.g., a ColE1-type and a p15A-type plasmid together). Step-by-Step Solution: Define incompatibility: inability of two plasmids with the same replicon system to be stably maintained together.Check choices: only the option describing two different plasmids failing to coexist without selection fits.Rule out host-restriction or conjugation-specific issues. Verification / Alternative check: Vector manuals often list the ori and Inc group to guide co-maintenance (e.g., pBR322 family vs. pACYC family) in a single host under dual selection if needed. Why Other Options Are Wrong: A: Host-range limitation is a separate concept from incompatibility. C: Both A and B are not necessarily true together. D/E: These do not define incompatibility; they are procedural outcomes. Common Pitfalls: Assuming antibiotic selection can “cure” incompatibility—selection masks but does not remove the underlying instability. Final Answer: Inability of two different plasmids to coexist stably in one host cell in the absence of selection

Question 4

Charon 34 and Charon 35 are examples of which type of cloning vectors derived from bacteriophage systems?

Accepted Answer

Introduction / Context: Historically important lambda vectors, such as the Charon series, enabled efficient cloning and library construction. Recognizing vector families by name is useful when reading older literature or troubleshooting legacy protocols. Given Data / Assumptions: Charon vectors are associated with bacteriophage lambda. They exploit lambda packaging and infection to deliver recombinant DNA into E. coli. They are not cosmids, plasmids, or modern BACs. Concept / Approach: Lambda vectors come in insertion and replacement forms, each with specific insert capacities and map features. The Charon series, like other lambda vectors, leverages headful packaging constraints and cos sites for efficient transduction and plaque formation to identify recombinants. Step-by-Step Solution: Match the proper names (Charon 34/35) to their vector family.Recall that Charons are classic lambda derivatives used for genomic libraries.Select “Lambda vectors.” Verification / Alternative check: Reference maps list Charon vectors with lambda arms, selectable markers, and sites engineered for replacement cloning strategies. Why Other Options Are Wrong: A: Plasmids replicate by ori-based replication; Charons rely on phage lambda life cycle features. B: Cosmids are plasmids with cos sites; Charon vectors are bona fide lambda phage derivatives. D: Phagemids are plasmids with filamentous phage origins, not lambda. E: BACs are large plasmid systems based on F-factor replicons. Common Pitfalls: Confusing cosmids (cos-containing plasmids) with clear lambda phage vectors due to the shared “cos” term. Final Answer: Lambda (phage λ) vectors

Question 5

Lambda gt10 vs. lambda gt11 — Which statement correctly distinguishes these two λ vectors used in cloning and expression?

Accepted Answer

Introduction / Context: Classic λ vectors differ in purpose: some are optimized for insert capacity and library construction, while others enable expression screening. λ gt10 and λ gt11 exemplify this split, and recognizing their roles prevents misapplication during library screening or expression cloning. Given Data / Assumptions: λ gt11 carries lacZ elements to permit expression of inserted sequences as fusion proteins. λ gt10 is used primarily for cloning (replacement) rather than expression screening. Both are lambda phage derivatives, not plasmids or cosmids. Concept / Approach: Expression vectors (like λ gt11) allow antigenic screening of plaques using antibodies against expressed fusion proteins. Replacement vectors (like λ gt10) maximize insert capacity by replacing dispensable lambda regions with foreign DNA, facilitating genomic library construction without necessarily expressing inserts. Step-by-Step Solution: Identify which vector supports lacZ fusion expression: λ gt11.Identify which vector is mainly for cloning capacity: λ gt10.Choose the option that states both parts correctly. Verification / Alternative check: Historic manuals for expression screening protocols (immunoscreening) reference λ gt11 specifically; mapping guides categorize λ gt10 as a high-capacity cloning vector. Why Other Options Are Wrong: B/D: Invert or deny expression capability of λ gt11. C: Neither is a cosmid; both are phage λ derivatives. E: Blue–white screening is a plasmid/phagemid trait (e.g., pUC/M13), not the primary feature of λ gt10. Common Pitfalls: Assuming any lacZ-related screening equates to blue–white colony selection; plaque immunoscreening in λ gt11 is a different methodology. Final Answer: λ gt11 is an expression vector that enables lacZ fusion expression, whereas λ gt10 is a cloning (replacement) vector

Question 6

The plasmid pBR322 carries which antibiotic resistance marker genes that are routinely used for selection and insertional inactivation?

Accepted Answer

Introduction / Context: pBR322 is among the most historically important E. coli plasmid vectors. Knowing its selectable markers is foundational for understanding classic cloning strategies, especially insertional inactivation screens using the Amp^r and Tet^r loci. Given Data / Assumptions: pBR322 contains bla (Amp^r) and tet (Tet^r) genes. Multiple cloning sites can disrupt Tet^r for insertional inactivation. Selection often starts with Amp^r, followed by replica plating to test Tet^r. Concept / Approach: Transformants are selected on ampicillin plates (Amp^r). Recombinants with inserts in the tet region lose tetracycline resistance, allowing discrimination between recombinant and non-recombinant clones via replica plating. This dual-marker system illustrates early cloning logic before modern blue–white screening became dominant. Step-by-Step Solution: Recall the two resistance markers on pBR322.Match them to the options.Select the answer listing both Amp^r and Tet^r. Verification / Alternative check: Standard plasmid maps confirm bla and tet loci on pBR322, with defined restriction sites used historically for insertional inactivation. Why Other Options Are Wrong: A/B/D/E: Each lists only one or a different marker set, not matching the canonical pBR322 features. Common Pitfalls: Confusing pBR322 with pUC plasmids, which carry Amp^r and lacZα for blue–white screening rather than Tet^r. Final Answer: Both ampicillin resistance (Amp^r) and tetracycline resistance (Tet^r)

Question 7

Phagemid vectors — What is the most accurate description of their structure and functional advantage in cloning workflows?

Accepted Answer

Introduction / Context: Phagemids merge convenient plasmid features (ori, antibiotic resistance, MCS) with a filamentous phage origin of replication (f1/M13). This enables recovery of single-stranded DNA after helper phage infection—critical for Sanger sequencing, mutagenesis, and phage display scaffolds. Given Data / Assumptions: Phagemids are primarily plasmids in growth behavior. An f1/M13 origin permits packaging of generated ssDNA into filamentous phage particles when a helper phage is present. They are distinct from lambda, cosmid, and BAC systems. Concept / Approach: A phagemid replicates as a double-stranded plasmid under normal growth. Upon superinfection with a helper phage, the f1 ori directs production of circular ssDNA that is secreted in filamentous particles. This duality combines ease of manipulation with the sequencing advantages of ssDNA templates. Step-by-Step Solution: Identify key hallmark: f1/M13 origin on a plasmid backbone.Relate to function: helper phage rescue produces ssDNA for downstream applications.Choose the option that captures both structure and function. Verification / Alternative check: Vector manuals for pBluescript, pGEM, and similar phagemids describe helper phage rescue protocols to obtain ssDNA for Sanger sequencing or mutagenesis. Why Other Options Are Wrong: B: Describes lambda-based systems, not phagemids. C: Incorrect biology; phages do not replicate as plasmids in eukaryotes. D: Cosmids lack the f1 origin and are not phagemids. E: Phagemids replicate autonomously in bacteria via plasmid ori; phage packaging is optional. Common Pitfalls: Confusing cosmid packaging (dsDNA) with filamentous phage ssDNA rescue; they serve very different purposes. Final Answer: Hybrid plasmids carrying a filamentous phage origin (f1/M13) to allow single-stranded DNA rescue

Question 8

Insert capacity question: λ gt10 and λ gt11 lambda vectors typically accommodate cloned DNA fragments up to about what size range?

Accepted Answer

Introduction / Context: Different λ vectors have different insert capacities depending on whether they are insertion or replacement types. λ gt10 and λ gt11 are classic insertion/expression vectors with modest capacities, appropriate for cDNA clones and short genomic fragments rather than very large inserts. Given Data / Assumptions: λ insertion vectors carry additional sequences, limiting insert size. Replacement vectors (other λ types) can accept larger inserts by replacing dispensable regions. Physical packaging constraints of lambda heads set upper and lower genome size limits for viable phage production. Concept / Approach: Viable lambda particles require a genome within a defined size window. Insertion vectors like λ gt10/11 allow only several kilobases of foreign DNA (commonly about 6–7 kb, sometimes up to ~8–10 kb depending on the build), whereas replacement vectors can accept larger fragments up to roughly 20 kb or more. Step-by-Step Solution: Recall that λ gt10/11 are not high-capacity replacement vectors.Match to typical insertion vector capacity: around 6–7 kb.Eliminate ranges that are either too small or indicative of BAC/cosmid capacities. Verification / Alternative check: Historic manuals and vector maps list capacities for λ gt10/11 in the mid–single-digit kilobase range for reliable cloning and packaging. Why Other Options Are Wrong: B: Too small; standard plasmids handle >1–2 kb easily. C/D: Reflect cosmid or λ replacement capacities, not λ gt10/11 insertion vectors. E: ≥100 kb indicates BAC/YAC scales, not lambda. Common Pitfalls: Assuming all λ vectors accept very large inserts; capacity depends on vector architecture. Final Answer: 6–7 kb

Question 9

Cosmid vectors are best suited for which general cloning task based on their hybrid plasmid–lambda design and packaging strategy?

Accepted Answer

Introduction / Context: Cosmids combine a plasmid backbone with lambda cos sites, enabling in vitro packaging into lambda heads for efficient transduction and selection. Their primary value lies in accommodating larger inserts than typical plasmids—bridging the gap between plasmids and BAC/YAC systems. Given Data / Assumptions: Cosmids retain plasmid replication and selection markers. Presence of cos sites allows lambda headful packaging. Insert sizes of roughly 35–45 kb are common in cosmid libraries. Concept / Approach: Lambda packaging imposes genome size constraints; cosmids exploit these to efficiently deliver large inserts while maintaining plasmid-like maintenance in E. coli. This makes them well-suited for intermediate-scale genomic library construction from both prokaryotic and eukaryotic sources. Step-by-Step Solution: Identify typical cosmid insert size range.Relate to lambda packaging via cos sites.Select the option describing large-fragment cloning using cosmid vectors. Verification / Alternative check: Genomic library protocols frequently specify cosmid vectors for 35–45 kb inserts, enabling robust coverage without moving to BAC/YAC complexity. Why Other Options Are Wrong: A: Too small; plasmids suffice for small fragments. C/D: Cosmids are agnostic to source; both prokaryotic and eukaryotic DNA can be cloned. E: Cosmids are not primarily expression tools; they are for large-fragment cloning. Common Pitfalls: Confusing cosmid capacity with lambda replacement vectors or BACs; cosmids occupy the middle ground. Final Answer: Cloning large DNA fragments (approximately 35–45 kb) using lambda packaging

Question 10

The λ ZAP system from Stratagene/Agilent is most appropriately categorized as which type of vector in its original packaged form?

Accepted Answer

Introduction / Context: λ ZAP is a specialized lambda vector system enabling rapid conversion of a recombinant phage clone into a plasmid (e.g., pBluescript) within the host using helper phage–mediated in vivo excision. Recognizing its starting category and special capability avoids classification errors. Given Data / Assumptions: Initial library construction and screening occur in a lambda phage context. In vivo excision generates a circular plasmid containing the insert for easy propagation. The system is not a cosmid or pure phagemid at the outset. Concept / Approach: By embedding a plasmid sequence inside a lambda vector, λ ZAP combines phage-based library screening with plasmid-level convenience. After identifying a positive plaque, excision yields a high-copy plasmid suited for restriction mapping, sequencing, and subcloning without re-cloning steps. Step-by-Step Solution: Identify original packaged form: lambda phage.Identify key feature: in vivo excision to plasmid (e.g., pBluescript SK).Choose the option that names lambda phage with excision capability. Verification / Alternative check: Vendor manuals describe λ ZAP as a lambda vector with excisable plasmid arms, not as a cosmid or basic phagemid. Why Other Options Are Wrong: B: While excision yields a plasmid related to phagemid features, the starting vector is lambda phage. C: Insert capacities and architecture differ from cosmids. D/E: Do not describe the lambda-based excision system. Common Pitfalls: Assuming that because the end product is a plasmid, the starting vector must be a plasmid or phagemid; λ ZAP starts as lambda. Final Answer: Lambda phage vector that allows in vivo excision to a plasmid

Question 11

Key feature of cosmids: Which essential lambda functions do cosmids intentionally lack while retaining cos sites and plasmid elements?

Accepted Answer

Introduction / Context: Cosmids are engineered to harness lambda packaging efficiency without carrying the full lambda genome. Understanding what they lack clarifies why cosmids behave as plasmids inside bacteria yet are packaged like lambda during transduction. Given Data / Assumptions: Cosmids include cos sites to enable in vitro lambda packaging. They retain plasmid ori and selection markers for propagation in E. coli. They do not encode the majority of lambda proteins needed for a viral life cycle. Concept / Approach: By omitting most lambda genes, cosmids avoid phage replication and lytic growth while still benefiting from headful packaging. After transduction, the cosmid circularizes and replicates as a plasmid, carrying large DNA inserts efficiently without phage biology complications inside the host. Step-by-Step Solution: Identify the lambda features present: cos sites only.Identify what is intentionally missing: most viral protein-coding genes.Select the option that precisely states this absence. Verification / Alternative check: Cosmid maps show cos sequences flanking a plasmid backbone with ori and antibiotic resistance but no λ structural/replication cassettes. Why Other Options Are Wrong: B/C/D: Cosmids retain ori, markers, and MCS—these are core plasmid features. E: Cosmids do replicate autonomously in bacteria via their plasmid ori. Common Pitfalls: Assuming “cos” implies a full phage genome; cosmids only borrow packaging signals. Final Answer: Genes coding for viral structural and replication proteins (most λ genes are absent)

Question 12

Cloning vectors in genetic engineering: A plasmid is considered a suitable cloning vector when which set of properties is satisfied?

Accepted Answer

Introduction / Context: In molecular cloning, the choice of vector determines how efficiently foreign DNA can be inserted, propagated, and recovered. Plasmids are popular cloning vectors because they are small, circular DNA molecules that replicate independently of the bacterial chromosome. This question tests the core design features that make a plasmid an effective vector in a teaching or research laboratory. Given Data / Assumptions: The goal is to insert, maintain, and recover a foreign DNA fragment in a bacterial host. Typical operations include restriction digestion, ligation, transformation, selection, and plasmid purification. Stable replication and ease of handling are essential. Concept / Approach: Good vectors combine practical lab handling with genetic features: a selectable marker, a well-characterized origin of replication, and a multiple cloning site containing unique restriction sites. Additionally, insertion of DNA should not abolish plasmid replication; the replication origin and copy-number control elements must remain intact regardless of the insert. Step-by-Step Solution: Assess isolation: researchers must purify plasmids easily from cells to analyze inserts and sequence clones.Assess restriction map: a unique site allows one, clean insertion point; multiple unique sites in an MCS offer flexibility without fragmenting the vector.Assess replication stability: inserting foreign DNA must not interrupt ori or essential replication genes; otherwise colonies may form but plasmids will be unstable.Combine criteria: a vector meeting all three is considered suitable for cloning. Verification / Alternative check: Standard plasmids (for example, pBR322-derivatives, pUC-series) are widely used because they satisfy all these properties and have robust documentation and protocols. Why Other Options Are Wrong: Readily isolated only: Helpful, but insufficient without a unique cloning site and stable replication.Unique restriction site only: Without reliable replication and selection, cloning fails.Replication unaffected only: By itself, this does not guarantee practical usability.Only antibiotic resistance is sufficient: Selection alone cannot ensure cloning or stability. Common Pitfalls: Inserting DNA into essential vector loci (ori, marker) or using vectors with duplicated restriction sites that produce unwanted fragmenting during cloning. Final Answer: All of the above.

Question 13

Phagemids in cloning: Identify the statement that is NOT true for a phagemid (plasmid–filamentous phage hybrid) system.

Accepted Answer

Introduction / Context: Phagemids are versatile vectors that combine features of plasmids and filamentous phage (such as M13). They are widely used for phage display, site-directed mutagenesis, and production of single-stranded DNA. Understanding their core genetic elements helps distinguish them from λ phage–based vectors and standard plasmids. Given Data / Assumptions: Phagemids possess a plasmid ori for double-stranded replication in bacteria. They also include an f1 origin to enable single-stranded DNA rescue with a helper phage. Selectable markers (for example, ampicillin resistance) are usually present. Concept / Approach: The key to phagemid function is the f1 origin derived from filamentous phage, not λ. With a helper phage, phagemids can be packaged into filamentous phage particles, allowing purification of single-stranded DNA. They are not λ-based integration vectors and therefore do not require a λ att site (used by λ for site-specific integration into the E. coli chromosome). Step-by-Step Solution: Check origin composition → plasmid ori + f1 ori (true).Check propagation modes → plasmid state in standard hosts; phage particle packaging in helper strains (true).Evaluate λ att site claim → phagemids are not λ integration vectors; this is not true.Confirm selectable marker and ssDNA utility → common and accurate features. Verification / Alternative check: Phagemid manuals and classic vector maps (for example, pBluescript, pUC-derivatives with f1 ori) show the f1 origin and standard antibiotic markers; no λ att is present. Why Other Options Are Wrong (i.e., they are true statements): Plasmid + f1 origins: Core architecture enabling ds and ss forms.Dual propagation modes: Fundamental to helper-phage rescue.Selectable marker: Required for maintenance and selection.ssDNA recovery: A hallmark application of phagemids. Common Pitfalls: Confusing λ-based replacement/insertion vectors with filamentous phage–based phagemids; assuming all phage-related vectors need λ att sites. Final Answer: Phagemids routinely contain a λ att site for integration into the host chromosome.

Question 14

λ phage packaging constraints: What total DNA size can be packaged efficiently into a lambda phage head during in vitro packaging?

Accepted Answer

Introduction / Context: Lambda phage (λ) vectors are used to clone relatively large DNA fragments. However, the phage head imposes strict size constraints: DNA that is too small or too large will not be packaged efficiently. Recognizing these limits is essential when constructing lambda replacement or insertion libraries. Given Data / Assumptions: λ headful capacity sets upper and lower boundaries for DNA length packaged. Replacement vectors remove a stuffer fragment to accommodate larger inserts. Insertion vectors accept smaller inserts and maintain most λ genome sequences. Concept / Approach: The lambda head requires DNA within a specific size window (roughly mid-30s to low-50s kb). Too little DNA is not recognized properly by packaging machinery; too much DNA cannot be physically accommodated. Therefore, inserts must be sized such that the total recombinant genome fits the packaging window. Step-by-Step Solution: Identify usable size range → approximately 35–53 kb total DNA.Evaluate vector type → insertion vectors handle smaller inserts; replacement vectors accept larger inserts by removing stuffer DNA.Select the answer that explicitly captures the window (not a single fixed size). Verification / Alternative check: Protocols for λ library construction specify calculating total recombinant size to fall within the packaging limits for efficient in vitro packaging and titer recovery. Why Other Options Are Wrong: 50 kb exact only: Overly restrictive; the system tolerates a range.40–50 kb only: Too narrow; underestimates the window.Any size: Incorrect; packaging has strict limits.< 25 kb only: Too small for efficient packaging. Common Pitfalls: Ignoring total genome size after ligation; miscalculating stuffer removal; attempting to package constructs outside the viable window leading to no plaques. Final Answer: Approximately 35–53 kb (defined lower and upper packaging limits).

Question 15

Expression from λ gt11: How is an inserted DNA fragment typically expressed when cloned into the lambda gt11 vector?

Accepted Answer

Introduction / Context: λ gt11 is a classic expression cloning vector used to produce fusion proteins for immunoscreening. The vector is designed so that inserted DNA is fused in-frame with the lacZ gene, producing a β-galactosidase fusion polypeptide. This enables plaque immunoscreening with antibodies against the fusion partner. Given Data / Assumptions: λ gt11 contains the lac promoter and the 5’ portion of lacZ. Cloning in-frame into the multiple cloning site yields a LacZ–insert fusion. Fusion proteins can be detected with anti-β-galactosidase or insert-specific antibodies. Concept / Approach: The vector architecture forces expression as a β-galactosidase fusion if the insert is properly oriented and in-frame. This facilitates expression screening and identification of antigenic clones directly on plates. Step-by-Step Solution: Recognize vector design → lacZ-based fusion system.Determine expression product → β-galactosidase fusion protein with the cloned insert.Eliminate alternatives that do not match the vector mechanism (free or secreted proteins). Verification / Alternative check: Protocols for λ gt11 immunoscreening emphasize detecting LacZ–fusion antigens in plaques using specific antisera. Why Other Options Are Wrong: Free cytosolic or secreted protein: Not the default outcome of λ gt11 design.All of the above/membrane-anchored: Not supported by the vector's expression strategy. Common Pitfalls: Frame-shifted inserts that abolish fusion; cloning in the wrong orientation; using inappropriate host strains lacking necessary lac expression components. Final Answer: As a β-galactosidase fusion protein from the lacZ gene.

Question 16

Plasmid phenotypes: What is meant by a “cryptic plasmid” in bacteria?

Accepted Answer

Introduction / Context: Not all plasmids encode easily recognized traits like antibiotic resistance or toxin production. The term “cryptic plasmid” is used for plasmids that appear phenotypically silent—at least under the conditions typically tested—yet they may still carry genes with subtle, conditional, or unknown functions. Given Data / Assumptions: Phenotype is assessed in standard lab media and conditions. Some plasmid genes express only under stress or specific environments. Genetic content may be small or poorly characterized. Concept / Approach: “Cryptic” refers to the absence of recognizable phenotypic output, not the absence of genes. Such plasmids might encode maintenance functions or context-dependent traits that escape detection in routine assays. Step-by-Step Solution: Define cryptic plasmid → lacking obvious selectable phenotype.Contrast with common plasmids → many have antibiotic markers or metabolic cassettes that produce clear traits.Select the option stating “no obvious phenotypic trait.” Verification / Alternative check: Molecular surveys often find small plasmids in environmental isolates with unknown gene content and no overt phenotype, fitting the “cryptic” label. Why Other Options Are Wrong: Many traits simultaneously: opposite of cryptic.Exactly one visible phenotype: contradicts the definition.Always antibiotic resistance: many plasmids lack such markers naturally.Obligate integration: unrelated to the cryptic concept. Common Pitfalls: Assuming “no phenotype” equals “no function”; overlooking conditional gene expression. Final Answer: A plasmid that does not confer any obvious phenotypic trait under standard laboratory conditions.

Question 17

Lambda cloning systems: How do Charon vectors differ from EMBL lambda vectors in library construction workflows?

Accepted Answer

Introduction / Context: Lambda-based cloning vectors exist in several families, notably Charon and EMBL series. Practical differences in how the vectors are prepared and how inserts are accommodated affect library construction efficiency and laboratory handling. Given Data / Assumptions: Both are λ-based vectors used for genomic library construction. Replacement strategies remove a central stuffer fragment and ligate inserts between λ arms. Lab workflows vary in how arms are prepared and how multi-site cloning is enabled. Concept / Approach: Charon vectors classically require a preparative step to separate λ arms from the central fragment before ligation to inserts. EMBL vectors were engineered to simplify arm preparation and typically provide improved polylinker choices and streamlined protocols. Therefore, a notable distinction is the requirement for physical arm separation in Charon workflows. Step-by-Step Solution: Identify Charon workflow → arm preparation and separation required.Identify EMBL improvements → easier stuffer removal and broader cloning sites.Select the statement that uniquely specifies the Charon requirement (arm separation). Verification / Alternative check: Protocol guides for Charon vectors detail extraction of λ arms; EMBL vectors emphasize simplified cloning with extended MCS regions. Why Other Options Are Wrong: More extensive polylinkers in Charon: more characteristic of EMBL, not Charon.Both (a) and (b): incorrect because (a) is false.No separation required: contradicts established Charon protocols.Identical: overlooks practical differences. Common Pitfalls: Confusing the roles of replacement vs. insertion vectors and assuming all λ systems are handled identically. Final Answer: Charon systems require physical separation of λ arms from the central fragment during cloning.

Question 18

EMBL lambda replacement vectors: What insert size range can be accommodated by EMBL3 and EMBL4 lambda vectors?

Accepted Answer

Introduction / Context: EMBL3 and EMBL4 are classic lambda replacement vectors designed to accept large DNA inserts by removing a central nonessential “stuffer” fragment. Knowing their insert capacity is important for planning genomic library construction and ensuring efficient packaging. Given Data / Assumptions: Lambda packaging has size limits that define total recombinant genome length. Replacement vectors allow larger inserts than insertion vectors. EMBL3/EMBL4 are optimized for large genomic fragments. Concept / Approach: By replacing a stuffer region, EMBL vectors can accept inserts on the order of tens of kilobases while keeping the total DNA within the λ packaging window. Their advertised capacity commonly falls in the ~40 kb range, enabling robust genomic library coverage. Step-by-Step Solution: Recognize vector type → replacement (large inserts).Match to typical capacity → approximately 40–44 kb inserts.Eliminate smaller ranges that fit insertion vectors or plasmids. Verification / Alternative check: Library construction manuals specify that EMBL replacement vectors handle inserts near 40 kb to obtain high-titer packaged phage. Why Other Options Are Wrong: 6–7 kb or 1–2 kb: too small; typical of plasmid or insertion-vector scales.15–25 kb: under the capacity of EMBL replacement vectors.> 60 kb: exceeds λ packaging constraints. Common Pitfalls: Confusing total genome length with insert length; forgetting to account for the remaining λ arms when calculating packaging size. Final Answer: 40–44 kb.

Question 19

Cosmids and cos sites: Which statements correctly describe the cos site used in cosmid cloning and packaging?

Accepted Answer

Introduction / Context: Cosmids are hybrid vectors that include the λ cos site, allowing plasmid-sized constructs to be packaged into λ phage heads for efficient delivery. Understanding the structure and role of the cos site clarifies how large inserts can be cloned and transported like phage, yet maintained as plasmids in the host. Given Data / Assumptions: Cos sites are the cohesive ends used by λ phage during packaging and circularization. Cosmids carry a plasmid origin plus cos sequences. After infection, cosmids circularize to replicate as plasmids. Concept / Approach: The λ cos site is a 12-base cohesive end that mediates concatemer cleavage and subsequent circularization in the host. In cosmids, cos enables λ head packaging of large DNA constructs, improving library construction efficiency compared with direct transformation of large plasmids. Step-by-Step Solution: Identify cos features → 12-base cohesive overhangs.Define function → circularization and ligation after delivery into the host cell.Select the combined statement reflecting structure and function. Verification / Alternative check: Cosmid protocols highlight using λ extract for packaging and subsequent recovery of circular plasmids in E. coli, all reliant on the cos sites. Why Other Options Are Wrong: Cos only a restriction site: incorrect; its role is cohesive-end pairing and packaging.Cos functions only in plasmids: it originates from λ and operates in λ packaging. Common Pitfalls: Confusing cos with general restriction sites; underestimating how cos improves transformation efficiency for large constructs via packaging. Final Answer: Both (a) and (b) are correct.

Question 20

Insertion vectors (lambda): What is the maximum practical size of foreign DNA that can be cloned using a λ insertion vector?

Accepted Answer

Introduction / Context: Lambda vectors come in two major formats for cloning: insertion vectors and replacement vectors. Insertion vectors retain most of the λ genome and accept relatively small inserts. Replacement vectors remove a stuffer fragment to accommodate much larger inserts. Knowing these capacities is crucial when selecting the right vector for a given fragment size. Given Data / Assumptions: Packaging constraints require the total recombinant DNA to fit within the λ headful window. Insertion vectors accept small inserts; replacement vectors accept much larger inserts. Common insertion vectors (for example, λ gt10) typically handle inserts up to roughly the 7–10 kb range. Concept / Approach: Because insertion vectors keep most of the λ genome intact, only a limited additional size can be added while keeping the total within packaging limits. In practice, inserts much above ~10 kb are not suitable for insertion vectors and should be moved to replacement formats. Step-by-Step Solution: Confirm vector class → insertion type retains bulk of λ DNA.Apply packaging window → little room left for extra DNA.Select the approximate maximum → around 10 kb insert size. Verification / Alternative check: Cloning guides for λ insertion vectors recommend inserts in the vicinity of several kilobases, and commonly state inefficiency beyond ~10 kb, whereas replacement vectors target ~15–20+ kb up to ~40 kb. Why Other Options Are Wrong: 35 kb or 50 kb: typical of replacement or cosmid systems, not insertion vectors.18 kb: exceeds practical insertion-vector capacity.< 2 kb only: too restrictive; insertion vectors can clone several kb effectively. Common Pitfalls: Attempting large genomic fragments in insertion vectors and obtaining very low packaging or plaque yields. Final Answer: About 10 kb.

Cloning Vectors Questions