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The molecular cloning technique employs a vector to transfer DNA fragments (target genes) into recipient cells, serving as a self-replicating DNA molecule tool. Common vectors include plasmids, bacteriophages, viruses, etc. Natural plasmids are circular DNA molecules capable of autonomous replication outside the chromosome, often carrying genes, especially those related to antibiotic resistance, which can be transferred between different bacterial cells. Plasmids are mainly found in bacteria, archaea, and eukaryotes, with some appearing in special organelles such as chloroplasts and mitochondria in plants. Plasmids are not exclusively circular; linear plasmids have been discovered in organisms like Streptomyces coelicolor and Borrelia hermsii.
Due to their ability to carry genes and transmit them between different cells, plasmids are employed in molecular cloning techniques. The plasmid vectors used in molecular cloning are artificially modified from natural plasmids to suit laboratory operations, with bacterial vectors being commonly used. In this technique, a target gene fragment is recombined into the plasmid to form a recombinant gene or construct. The recombinant plasmid is then introduced into recipient cells (such as Escherichia coli) via microbial transformation techniques, allowing the target gene within the recombinant DNA to replicate or express in the recipient cells, thereby altering the host cell's characteristics or producing new substances. Bacterial plasmids are the most commonly used vectors in genetic engineering, and these plasmids must possess at least an origin of replication, a selection marker, and cloning sites.
The structure of bacterial plasmids.
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ORI is a DNA sequence serving as the starting point for plasmid replication. DNA helicase acts on this sequence, causing the double-stranded DNA to unwind and replication to commence. Plasmids must be replicable; otherwise, their numbers will quickly diminish as bacteria grow.
The antibiotic resistance gene serves for rapid selection. Bacteria containing the desired plasmid can grow in culture media containing antibiotics, while those without the plasmid are killed off.
A marker used to select cells that have successfully taken up the plasmid. This marker needs to be distinct from antibiotic resistance in bacteria. Bacterial resistance gene selection is used during plasmid amplification, while selectable markers are used to screen or label transfected cells. Selectable markers are typically antibiotic resistance or fluorescent proteins.
A DNA sequence that initiates gene transcription. Promoters are recognized by RNA polymerase, initiating transcription. Typically located upstream of the target gene within 100-1000 base pairs, promoters are vital components of plasmids, determining where the target gene can be expressed and the level of protein expression.
MCS is a short DNA segment containing multiple single sites for restriction enzymes, facilitating the insertion of foreign genes. Generally, the longer and more unstable the foreign DNA fragment, the harder it is to insert, leading to lower transformation efficiency. In expression plasmids, the MCS is often positioned downstream of the promoter.
A short DNA segment used for PCR amplification or sequencing of the plasmid.
Also known as an exogenous gene, it is the gene, promoter, or other DNA segment cloned into the MCS, typically representing the genetic elements researchers wish to study.
Used for gene expression (studying gene function). Expression vectors must contain a promoter, transcription termination sequence, and the insert gene. The promoter drives transcription of the inserted DNA to produce RNA, and the termination sequence on the synthesized RNA signals the end of transcription. Enhancer sequences can increase the production of protein or RNA. Expression vectors can drive expression in various cell types (mammalian, yeast, bacterial, etc.), largely depending on the promoter used to initiate transcription.
Used to facilitate the cloning of DNA fragments. Cloning vectors are often simple, typically containing bacterial resistance genes, an origin of replication, and a Multiple Cloning Site (MCS).
Used to study the function of genetic elements. These plasmids contain a reporter gene (e.g., luciferase or GFP), providing a readout of genetic element activity.
Effectively deliver genetic material to target cells through modified viral genomes. These plasmids can be used to produce viral particles, such as lentiviral, retroviral, AAV, or adenoviral particles, for efficient infection of target cells.
Used to reduce the expression of endogenous genes. This is achieved by expressing shRNA targeting the mRNA of the target gene. These plasmids have promoters that can drive the expression of short RNA.
Used for targeted genome editing. Typically accomplished using CRISPR technology.
This is the most common type of plasmid, capable of autonomous replication within cells. Self-replicating plasmids typically contain the origin of replication (ori) required for autonomous replication, as well as other necessary elements such as selectable markers and gene expression components.
These plasmids are widely used in RNA interference (RNAi) research. RNAi plasmids contain sequences that produce small interfering RNA (siRNA) or short hairpin RNA (shRNA). These RNA molecules can target specific gene mRNA and inhibit the expression of that gene. These plasmid types play important roles in molecular biology and genetic engineering research, used for gene cloning, gene expression, and gene silencing studies. Different plasmid types have different functions and applications, so selecting the appropriate plasmid type is crucial based on the research objectives.
Traditionally, plasmid recombination is based on the principle of complementary base pairing. The general procedure is as follows:
Gateway Cloning Technology utilizes proprietary recombination sequences to facilitate the more efficient insertion of DNA fragments into plasmids. It can be applied to cloning large gene fragments and enables DNA transfer between different expression vectors while maintaining the correct reading frame. This technology integrates two flanking recombination sequences, att L1 and att L2, at both ends of the inserted DNA fragment, creating a channel-like structure referred to as the "entry clone." This avoids issues with cutting points within the target fragment, maintaining the integrity of large DNA fragments and significantly improving cloning efficiency. It is commonly used for integrating large DNA fragments into the same expression vector and is thus termed high-throughput gene cloning technology.
Ligation-Independent Cloning (LIC) involves using linearized pET vectors with 12-15 protruding bases at the ends for annealing with complementary sequences of the target fragment during annealing. When designing PCR primers, sequences complementary to the LIC vector are added. The PCR product is digested with a 3' to 5' exonuclease to expose a single-stranded sequence complementary to the vector. This allows for efficient directional insertion of the target fragment into the LIC vector. Positive clones are typically first screened in bacterial strains lacking the DE3 segment and then transformed into expression strains such as BL21 (DE3) for induction with IPTG.
Subsequent to LIC, SLIC technology was developed, eliminating the principle of complementary base pairing and establishing a cloning method independent of sequence and connection. The steps are similar, with PCR amplification primers designed for the target DNA molecule, adding 20-30 bp DNA fragments homologous to the vector at both ends. The vector plasmid is linearized to expose homologous sequences; Escherichia coli transformation; and recombinant screening. Like the traditional plasmid recombination method, the restriction enzyme cleavage (restriction endonuclease reaction) and ligation (homologous recombination) processes of this technique are also carried out step by step.
Gibson Assembly technology is suitable for joining multiple linear DNA fragments and assembling vectors. The master mix contains three types of enzymes: an exonuclease that digests DNA from the 5' end, generating long sticky ends that facilitate pairing with other homologous ends; a polymerase for repairing base gaps; and a DNA ligase for seamless assembly, forming complete DNA molecules. In principle, Gibson Assembly continues the complementary base pairing principle of traditional plasmid recombination, but with longer complementary sequences. This reaction requires both restriction endonucleases and restriction exonucleases, and the restriction enzyme cleavage (exonuclease) and ligation reactions are completed in the same system, resulting in shorter reaction times and higher efficiency.
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