Horizontal gene transfer – where genes are transferred to another organism (host cell) that is not related to the donor cell – is often used in genetic engineering; this process is called transformation. The possibilities of molecular biology allowed scientists, to use artificially created plasmids to transfer foreign genetic material into cells. Plasmids are a form of organization of bacterial DNA — small double-stranded circular DNA molecules capable of autonomous replication. Naturally, bacteria plasmids usually contain "useful" genes, such as antibiotic resistance genes. In this topic, we are going to learn how to create artificial plasmids and transfer them to a cell so that it synthesizes the desired proteins.
Artificial plasmids structure and importance
One of the most important methods of genetic engineering is the introduction of foreign genes into a cell in order to obtain the target protein. But it is useless to introduce DNA into a cell just like that – the cell defends itself well and breaks down all foreign molecules. So the best way is to implement foreign DNA into a cell by special molecules called vectors (a special design designed to deliver DNA to a bacterium). Foreign DNA can then be inserted in the vector, and then the vector-DNA complex can be transferred into the cell (this process is called transfection). So, the main components of the transfection are:
1) A vector for introducing the target fragment into the host cell, most often plasmids.
2) Insert – a target DNA fragment, such as a gene, regulatory element, or operon.
Plasmid vectors are quite complex structures, and there are certain rules for their successful assembly. The main part of the vector is the site with the inserted gene that we want to express in the host cells to make some particular protein. In addition, the plasmid must have a site of replication, a promoter region (necessary for the cell to transcription process), and one multiple cloning site (MCS) that contains restriction sites for subsequent foreign DNA insertion. The last mandatory component of the vector is the antibiotic resistance gene (this insert is necessary for further selection and will be discussed later). There are sometimes regions in the plasmid that are different from those listed above. This usually depends on the goals of the experiments or the methods of assembling the vectors.
Restriction and ligation
In order to combine the plasmid and the desired gene, two types of enzymes are used: restriction enzyme (restictase) and DNA ligase. A restriction enzyme is an enzyme that cuts DNA and linearized it. It can only work with a specific target sequence. These sequences are found in multiple cloning site (MCS) and on this sequence, restictase makes the cut. At the output, depending on the type of enzyme used and the type of DNA, either
1) simple breaks are formed;
2) in the case when the plasmid MCS sequence and the gene sequence are complemented, short single-stranded ends (the so-called cohesive ends) are formed. These cohesive ends improve the efficiency of further crosslinking because they allow the gene to bind complementarity to the plasmid.
In order to crosslink the gene and the plasmid, the enzyme ligase is used. Generally, ligase is one of the most important enzymes in the cell life cycle. The main task of the ligase is to bring together fragments of newly synthesized DNA and form a complete chain without gaps. The same ability is used to create a recombinant plasmid: a ligase can connect them together to form a continuous molecule.
Using the energy of an ATP molecule, ligase catalyzes a reaction in which the phosphate group at the 5' end of one DNA strand binds to the hydroxyl group at the 3' end of another strand. As a result of this reaction, an integral sugar-phosphate backbone is formed.
Assembly diversity
Within the restriction site, there are many places where the enzyme cuts the plasmid. Then, after ligation, variants of the plasmid may arise that are not needed. For example, a cut plasmid can close on itself without inserting a gene. In addition, the gene could stand up incompletely or in the wrong direction. These and some other errors can lead to the appearance of recombinant plasmids without the target gene, which will not "work" in cells after transformation.
Therefore, after transformation, cells most often undergo primary selection. They are seeded on a medium with an antibiotic – this allows you to get only those colonies that have received the correct plasmid structure, which usually has a special site with an antibiotic resistance gene. In addition, recombinative selection is often carried out – selection of only those cells in which the target protein is synthesized: for example, by isolating a plasmid from a colony of bacteria and comparing its composition and structure with an ideal plasmid.
Plasmids creation process
So, the final general scheme of cell transformation is as follows:
1) Plasmid target DNA fragment selection: we can use restriction endonucleases, PCR reaction, or assembling from individual nucleotides.
2) The plasmid is then linearized with restriction enzymes.
3) The free cohesive ends of the plasmid and the insert gene are modified to allow their joining by DNA ligase, or, sometimes, in vivo repair mechanisms (the cell's own defense systems that stitch breaks in DNA).
4) The plasmid and target gene are then fused into one complex and form a recombination plasmid.
5) Plasmid is transformed into the host cell.
Conclusion
So, in order to introduce DNA into a bacterial cell and for the gene to start working, vectors are used. Usually, these are plasmids, circular small DNA. First, the plasmid is cut with a restriction enzyme, after which the ligase enzyme crosslinks the gene and the plasmid. After that, a transformation takes place. Sometimes errors occur during the creation of a recombinant plasmid or during a transformation so that after the seeding of bacterial colonies, they are selected.