Plasmids are small circular double-stranded DNA molecules found in many microorganisms (especially bacteria) that can replicate independently of chromosomal DNA. Artificially engineered DNA plasmid vectors are among the most widely used tools in modern molecular biology and genetic engineering. They are applied to gene function analysis, protein expression, cell tracing, and a variety of diagnostic and screening experiments.
I. Basic Concept of Plasmids
A plasmid is a small, circular, double-stranded supercoiled DNA molecule that can replicate independently using the host cell’s replication machinery without integrating into the host chromosome. After artificial modification, it can carry specific genes of interest along with regulatory and selection elements, and is therefore commonly referred to as a DNA vector (also called a cloning/expression vector). In molecular cloning and recombinant DNA technology, its core functions include carrying and stably maintaining the gene of interest sequence, mediating transcription and translation of the gene of interest in host cells, selecting successfully transformed cells via selection markers, and controlling plasmid copy number through the origin of replication to meet experimental needs such as protein expression, reporter gene detection, or large-scale plasmid DNA preparation.
II. Overview of the Five Key Structural Modules of Plasmids
A typical experimental plasmid usually contains the following five core structural modules:
1.Gene of interest
2.Selection marker, such as an antibiotic resistance gene
3.Origin of replication (origin of replication, Ori)
4.Transcription promoter (promoter)
5.Multiple cloning site (multiple cloning site, MCS)
Figure 1 Core elements of a DNA plasmid (gray circle) include: the gene of interest (Gene X), selection marker (AmpR), origin of replication (Ori), transcription promoter (Promoter), and multiple cloning site (MCS).
III. Gene of Interest
The primary function of a plasmid is to serve as a carrier for the gene of interest (a specific gene sequence that researchers aim to express, regulate, or functionally analyze in host cells). Applications include purifying the recombinant protein encoded by the gene for structural and functional studies, conferring new phenotypes or functions to cells (e.g., expression of fluorescent proteins or drug-resistance–related proteins), and serving as a reporter system to monitor upstream regulatory element activity. For example, plasmids containing a luciferase gene are often used as reporter vectors; quantifying luciferase activity enables analysis of promoter activity and monitoring of gene expression levels, thereby indirectly reflecting the regulatory effects of specific cis-regulatory elements or signaling pathways.
IV. Selection Marker
Selection markers are another indispensable class of functional genes on plasmids, used to distinguish host cells that carry the plasmid from those that do not. The basic principle is that only cells containing the plasmid and expressing the selection marker gene can survive or proliferate under specific selection conditions.
1.Antibiotic resistance markers in bacteria
Plasmid selection markers are host-specific. In commonly used bacterial plasmid systems, selection markers are mostly antibiotic resistance genes. For instance, AmpR confers resistance to ampicillin; other common types include kanamycin resistance genes. Successfully transformed cells carrying the plasmid can grow normally on media containing the corresponding antibiotic, whereas untransformed cells are sensitive and cannot form colonies, enabling positive clone selection.
2.Nutritional auxotrophy complementation markers in eukaryotic microorganisms such as yeast
In eukaryotic microorganisms such as Saccharomyces cerevisiae, selection is often achieved by complementation of nutritional auxotrophy. For example, plasmids carry genes encoding key enzymes in amino acid or nucleotide biosynthesis pathways to complement the corresponding defects in host strains, allowing only plasmid-bearing cells to grow on selective media lacking the nutrient. Although selection strategies differ across hosts, the core principle is to restrict growth to the cell population carrying the plasmid.
V. Origin of Replication (Ori)
The origin of replication is a specific DNA sequence on the plasmid recognized and assembled by the host replication machinery, forming the basis for plasmid maintenance and amplification in cells.
1.Ori and DNA replication
Origins of replication are present in both eukaryotic and prokaryotic chromosomal DNA. On plasmids, the Ori can be recognized by host proteins to initiate plasmid DNA replication, ensuring that plasmids are stably partitioned into daughter cells during cell division.
2.Ori, plasmid copy number, and applications
Different types of origins determine plasmid copy number in host cells. Copy number is closely linked to experimental applications—high-copy plasmids are suitable for high-level expression of target proteins or reporter genes, or for producing large amounts of plasmid DNA. Low-copy plasmids are better for expressing toxic proteins or conducting finely controlled functional studies (reducing the burden on host cell growth). Therefore, when designing or selecting plasmid backbones, it is necessary to choose vectors with an appropriate Ori according to experimental goals.
VI. Transcription Promoter
A promoter is a cis-acting element upstream of the gene of interest and a key region recognized and bound by RNA polymerase. It determines the transcription start site and transcription efficiency of the gene of interest.
1.Promoters and transcriptional regulation
In plasmid vectors, promoter choice directly determines the transcription level and expression pattern of the gene of interest. Strong promoters drive high-level transcription and are suitable for large-scale protein expression or high-sensitivity reporter detection. Weak promoters are appropriate for proteins that are toxic or highly perturbing to host cells, to avoid overexpression-induced death or severe stress. Inducible promoters initiate transcription only under specific induction conditions or in the presence of inducers, enabling temporally and dose-controlled gene expression. Literature (Vandierendonck et al., 2023) indicates that when expressing cytotoxic proteins, using weaker or controllable expression systems can improve experimental success rates and yield interpretable biological phenomena.
2.Promoter integrity and plasmid function
As an essential element for transcription initiation, point mutations, deletions, or insertions within promoter regions may prevent effective RNA polymerase binding, significantly reducing or completely suppressing gene expression. Therefore, when experiments show that “the plasmid is present but protein/reporter signals are absent or markedly reduced,” promoter regions should be sequenced or functionally validated to rule out potential mutations.
VII. Multiple Cloning Site (MCS)
The multiple cloning site is a short, artificially designed DNA segment containing multiple different restriction endonuclease recognition sites. Its main role is to provide flexible and diverse restriction site combinations for inserting or replacing the gene of interest.
1.Traditional restriction enzyme cloning
Traditional restriction enzyme cloning relies on restriction endonucleases that recognize specific short sequences to cut DNA. One or more enzyme pairs are used to cut the plasmid around the MCS to remove the original insert. After purification of the cut backbone, DNA ligase catalyzes phosphodiester bond formation to ligate the gene of interest fragment—bearing compatible restriction ends or overlapping regions—into the plasmid backbone. The ligation product is then transformed into host cells and positive clones are selected. Because the MCS contains a cluster of commonly used restriction sites, enzyme strategies can be chosen flexibly based on insert characteristics and sequence composition.
2.Relationship between modern seamless cloning and the MCS
With advances in molecular cloning, seamless strategies such as Type IIS restriction enzyme–based modular assembly and homologous recombination–mediated cloning have become increasingly common. These methods do not depend on classic restriction sites within traditional MCS regions, but instead rely on specific digestion systems or longer homologous overlaps for fragment assembly. As a result, the importance of a traditional MCS is relatively reduced. Nevertheless, in many existing vector systems, the MCS remains an important reference region for evaluating plasmid structural rationality and operability.
VIII. Aladdin-Related Products
Reagent | CAS No. | Plasmid-related application scenarios |
Tetracycline Hydrochloride | Corresponds to the tetᵣ resistance gene; used for construction and selection of bacterial clones harboring tetracycline-resistant plasmids. | |
Chloramphenicol | Corresponds to the cat chloramphenicol resistance gene; used for plasmid selection and stable maintenance (especially common in low-copy plasmid systems). | |
RNase A | Specifically degrades host RNA during plasmid extraction to prevent RNA contamination; commonly used with lysis or neutralization buffers at a final concentration of ~20 μg/mL. | |
Calcium Chloride | Used to prepare chemically competent cells (e.g., DH5α); Ca²⁺ increases membrane permeability and, together with ice incubation + heat shock, enables plasmid transformation. | |
Isopropanol | Used to precipitate DNA during plasmid extraction; can be performed at room temperature, precipitates quickly, and helps reduce some RNA impurities. | |
Glycerol | Used for long-term plasmid storage and preparation of glycerol stocks (final concentration 15%–20%) to protect plasmids and cells from freeze–thaw damage; also used for competent-cell freezing. | |
Agarose | Used to make agarose gels for plasmid electrophoretic verification and quality assessment; distinguishes supercoiled, nicked/open-circular, and linear plasmids, and size differences between recombinant plasmids and empty vectors. | |
Tris Base (Tris(hydroxymethyl)aminomethane) | A key component of many plasmid-related buffers (e.g., TAE/TBE, running buffers, lysis/neutralization buffers, storage buffers); maintains pH stability and improves plasmid DNA stability. | |
Ethidium Bromide | Classic intercalating nucleic-acid dye that fluoresces under UV when bound to dsDNA; used to visualize plasmid bands and conformations (supercoiled/linear/open-circular) in agarose gels. | |
IPTG (Isopropyl β-D-thiogalactopyranoside) | A lactose analog that induces lac operon/promoter-driven expression of target or reporter genes; often used with plasmids containing lacO/MCS elements (e.g., lacZα fragment). | |
X-Gal (5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside) | Used with IPTG for blue-white screening in the lacZα complementation system; directly linked to whether MCS insertion disrupts the lacZα fragment. | |
L-Arabinose | Induces PBAD promoter-controlled plasmid expression systems, typical for plasmids containing araBAD regulatory elements; enables fine control of gene expression by modulating promoter activity. |