Introduction

Plasmid DNA is essential to genetic engineering due to its role in molecular cloning and production of bioengineered products, such as therapeutic proteins. Here, we explore the role of plasmid vectors and recombinant DNA in the plasmid DNA development workflow, covering their design, construction and use in gene cloning, gene amplification, and transfection into cells.

What is A Plasmid?

Plasmids were first found in bacteria as extrachromosomal genetic material. Plasmids are small, circular arrangements of DNA that are not integrated into an organism’s genome and can replicate independently of genomic DNA.

Plasmid DNA is mainly found in bacteria, carrying genes that serve survival functions, such as antibiotic resistance and rapid replication. Plasmids have been discovered in yeasts and plants, as well as organelles of eukaryotic organisms.

Plasmids as Vectors in Genetic Engineering Applications

Finding an ideal vector for genetic engineering is integral to successfully transferring foreign genetic material into another organism and rapidly replicating it without degradation by the host. Plasmids offer several advantages, lending them to widespread use in biotechnology and genetic engineering.

Their relatively small backbone size makes them ideal for vector isolation and purification, while the circular structure makes them more stable and resistant to degradation than linear DNA. In addition, cloning plasmids can harbor high copy numbers within a host that can lead to large numbers of extractable plasmids.

The gene of interest is amplified with primers and inserted into a plasmid by annealing to complementary sequences at a restriction site and transformed into a host, where the plasmid origin of replication allows the inserted gene and plasmid vector to be self-replicated separately from the host's chromosomal DNA. Other plasmid genes, particularly those responsible for antibiotic resistance or fluorescence, can serve as markers that help identify cells with successful plasmid intake.

Artificial plasmids can be used as vectors for recombinant protein production or gene therapy creation. They can facilitate the production of proteins such as therapeutic antibodies or antibody-drug conjugates, treating diseases such as cancer or autoimmune diseases. For example, plasmids are commonly employed to bioengineer E. coli or S. cerevisiae for large-scale insulin production for diabetes patients. In addition, preclinical and clinical studies with plasmid DNA have returned promising results pointing to potential applications in gene therapy. In developing gene therapies for HER2-overexpressing cancers, plasmid DNA was used to encode anti-HER2 monoclonal antibodies, which reversed tumor growth in human muscle cell lines and mice models. In a phase I study enrolling seven patients, the overexpression of integrin that accelerated angiogenesis and metastasis in solid tumors was counteracted by the delivery of a plasmid DNA that encoded an integrin inhibitor peptide.

Plasmid Structure

A plasmid DNA construct must contain essential sequences to exert its function.

1. Origin of replication (ORI) sequences for autonomous plasmid DNA replication.
2. Antibiotic resistance gene for selection of transfected cells on a selective media.
3. Restriction sites, also known as multicloning sites, for foreign DNA insertion using restriction enzymes.
4. Promoter Region for initiating target (gene of interest) DNA transcription.

Construction of the Plasmid DNA Cloning Vector

Plasmid construction involves combining a complete plasmid vector backbone for genetic engineering with a foreign gene of interest, inserting the foreign DNA fragment into the vector. First, a DNA fragment carrying the gene of interest is isolated from its genome, while the plasmid is harvested and purified as described below.

Plasmid backbones contains restriction sites. Restriction enzymes cut the plasmid DNA and the PCR-amplified genes at their corresponding restriction sites, creating complementary sticky ends that are mixed in a solution. Finally, DNA ligase is added to the solution to glue the target gene and plasmid DNA together, forming a double-stranded circular DNA.

Transforming Plasmid DNA into the Host Cell

After plasmid DNA and the target gene are ligated, the plasmid construct must be introduced into bacterial cultures, such as E. coli, which rapidly replicate and be produce the plasmid on a large scale. The process of transformation involves increasing the permeability of bacterial cell walls and membranes to allow for the sufficient uptake of the plasmid construct.

Common transformation methods permeabilize cell membranes and can be achieved using heat shock, and electroporation. Bacterial cultures are treated with calcium chloride (CaCl2) to make them amenable to transformation. Cultures are chilled near 0oC and exposed them to heat shock near 30oC , which creates transient pores throughout the membrane for plasmid DNA entry. In electroporation, membrane permeability is induced by exposing cultures to electric currents that create transient membrane pores. Beyond electroporation and heat shock, other techniques for difficult vectors or strains include microinjection for the direct transfer of plasmid DNA, using bacterial conjugation to horizontally transfer the plasmid from one bacterial strain cell to another, and liposome-mediated gene transfer.

Selection

Following transformation, the bacteria culture is grown on a selective media containing an antibiotic to select for the cells carrying the plasmid construct and kill cells not carrying the plasmid. The antibiotic resistance gene in the backbone of the plasmid is key to this selection. Antibiotics are added to the culture media or individual plates, ensuring that only the cells carrying the plasmid construct with the resistant gene survive.

Finally, the isolation and purification techniques used to obtain the initial pure plasmid DNA are employed again to harvest the plasmid constructs from the bacteria culture.

Plasmid DNA Isolation

Before plasmid DNA can be used to for protein production, it must be harvested and separated from the bacterial chromosomal DNA.

There are a multiple lysis methods used to harvest plasmid DNA. One of the most common techniques is Alkaline lysis, performed by first pelleting the bacterial cells in a centrifuge and suspending them in a basic solution with sodium hydroxide (NaOH) and Sodium Dodecyl Sulfate (SDS) detergent buffer. This solution helps break bacterial cell membranes and cell walls.

Purification of the Plasmid DNA

DNA, including plasmid DNA and genomic DNA, must be obtained from the solution by eliminating residual proteins and contaminants. Several methods exist for this purpose.

1. Phenol-chloroform mixtures help remove proteins and lipids, separating plasmid DNA from cellular debris.
2. Spin columns pass the plasmid DNA solution through a solid matrix that selectively binds nucleic acids. Proteins, lipids and salts are washed away in the process.
3. Plasmid DNA, like other nucleic acids, is insoluble in ethanol solution. Ethanol induces plasmid DNA precipitation, which can be extracted from cellular debris upon centrifugation.
4. Magnetic beads can bind plasmid DNA in a solution and separate it from contaminants.

After purification, plasmids are separated from genomic DNA by selecting for the smaller size of the plasmid DNA relative to genomic DNA. Techniques such as gel electrophoresis, ultracentrifugation, and anion exchange chromatography can be used. Screening methods such as sanger sequencing or NGS are required ensure plasmids have not mutated and retain the correct gene insertion. This step is crucial before the downstream applications in gene therapy and protein production. Our article DNA Sequencing for Plasmids covers the strategies for characterizing the plasmid construct.Screened plasmids are cell banked and can be transformed into protein production bacterial strains for expression of bioengineered products.

Choosing the Ideal Plasmid Type as a Cloning Vector

The choice of cloning vector depends on the size of the DNA fragment to insert. In general, plasmid DNA is suitable for inserting fragments of up to 15 kbp.

Special vectors beyond plasmids are preferable when incorporating larger fragments. More importantly, they are used to generate genomic libraries employed in whole-genome sequencing of organisms for genetic engineering applications.

1. Cosmid is a special plasmid carrying bacteriophage Cos sites that allow the packaging of the gene into bacteriophage λ particles, increasing the insertion capacity up to 45 kbp.
2. Fosmid is derived from bacterial F-plasmids, which comprise distinct gene partitioning mechanisms for intercellular gene transformation. Their insert gene size is up to 40 kbp. Fosmids are advantageous for their low copy numbers, which improves the stability of the cloned DNA fragments.
3. Bacterial artificial chromosomes are derived from F-plasmids that allow the insertion of genes between 150-350 kbp. They have been used extensively for whole genome sequencing in the Human Genome Project.

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