Recall that these mechanisms are examples of horizontal gene transfer —the transfer of genetic material between cells of the same generation. Figure 1. In this example, the human insulin gene is inserted into a bacterial plasmid. This recombinant plasmid can then be used to transform bacteria, which gain the ability to produce the insulin protein.
Herbert Boyer and Stanley Cohen first demonstrated the complete molecular cloning process in when they successfully cloned genes from the African clawed frog Xenopus laevis into a bacterial plasmid that was then introduced into the bacterial host Escherichia coli. Molecular cloning is a set of methods used to construct recombinant DNA and incorporate it into a host organism; it makes use of a number of molecular tools that are derived from microorganisms. In recombinant DNA technology, DNA molecules are manipulated using naturally occurring enzymes derived mainly from bacteria and viruses.
The creation of recombinant DNA molecules is possible due to the use of naturally occurring restriction endonucleases restriction enzymes , bacterial enzymes produced as a protection mechanism to cut and destroy foreign cytoplasmic DNA that is most commonly a result of bacteriophage infection. Stewart Linn and Werner Arber discovered restriction enzymes in their s studies of how E. Today, we use restriction enzymes extensively for cutting DNA fragments that can then be spliced into another DNA molecule to form recombinant molecules.
Each restriction enzyme cuts DNA at a characteristic recognition site , a specific, usually palindromic, DNA sequence typically between four to six base pairs in length. A palindrome is a sequence of letters that reads the same forward as backward. A restriction enzyme recognizes the DNA palindrome and cuts each backbone at identical positions in the palindrome. Some restriction enzymes cut to produce molecules that have complementary overhangs sticky ends while others cut without generating such overhangs, instead producing blunt ends Figure 2.
Molecules with complementary sticky ends can easily anneal , or form hydrogen bonds between complementary bases, at their sticky ends. The annealing step allows hybridization of the single-stranded overhangs. Hybridization refers to the joining together of two complementary single strands of DNA.
Blunt ends can also attach together, but less efficiently than sticky ends due to the lack of complementary overhangs facilitating the process. In either case, ligation by DNA ligase can then rejoin the two sugar-phosphate backbones of the DNA through covalent bonding, making the molecule a continuous double strand. Figure 2. This is known as a palindrome. The cutting of the DNA by the restriction enzyme at the sites indicated by the black arrows produces DNA fragments with sticky ends.
Another piece of DNA cut with the same restriction enzyme could attach to one of these sticky ends, forming a recombinant DNA molecule. After restriction digestion, genes of interest are commonly inserted into plasmids , small pieces of typically circular, double-stranded DNA that replicate independently of the bacterial chromosome see Unique Characteristics of Prokaryotic Cells.
Plasmids used as vectors can be genetically engineered by researchers and scientific supply companies to have specialized properties, as illustrated by the commonly used plasmid vector pUC19 Figure 3. Some plasmid vectors contain genes that confer antibiotic resistance ; these resistance genes allow researchers to easily find plasmid-containing colonies by plating them on media containing the corresponding antibiotic.
The antibiotic kills all host cells that do not harbor the desired plasmid vector, but those that contain the vector are able to survive and grow. Figure 3. Arrows indicate the directions in which the genes are transcribed. Note the polylinker site, containing multiple unique restriction enzyme recognition sites, found within the lacZ reporter gene. Also note the ampicillin amp resistance gene encoded on the plasmid. Plasmid vectors used for cloning typically have a polylinker site , or multiple cloning site MCS.
A polylinker site is a short sequence containing multiple unique restriction enzyme recognition sites that are used for inserting DNA into the plasmid after restriction digestion of both the DNA and the plasmid. Having these multiple restriction enzyme recognition sites within the polylinker site makes the plasmid vector versatile, so it can be used for many different cloning experiments involving different restriction enzymes.
This polylinker site is often found within a reporter gene , another gene sequence artificially engineered into the plasmid that encodes a protein that allows for visualization of DNA insertion.
The reporter gene allows a researcher to distinguish host cells that contain recombinant plasmids with cloned DNA fragments from host cells that only contain the non-recombinant plasmid vector. The most common reporter gene used in plasmid vectors is the bacterial lacZ gene encoding beta-galactosidase, an enzyme that naturally degrades lactose but can also degrade a colorless synthetic analog X-gal , thereby producing blue colonies on X-gal—containing media.
The lacZ reporter gene is disabled when the recombinant DNA is spliced into the plasmid. Because the LacZ protein is not produced when the gene is disabled, X-gal is not degraded and white colonies are produced, which can then be isolated. This blue-white screening method is described later and shown in Figure 4.
In addition to these features, some plasmids come pre-digested and with an enzyme linked to the linearized plasmid to aid in ligation after the insertion of foreign DNA fragments. Figure 4. Click for a larger image.
The steps involved in molecular cloning using bacterial transformation are outlined in this graphic flowchart. The most commonly used mechanism for introducing engineered plasmids into a bacterial cell is transformation , a process in which bacteria take up free DNA from their surroundings. Some bacteria, such as Bacillus spp. However, not all bacteria are naturally competent. In most cases, bacteria must be made artificially competent in the laboratory by increasing the permeability of the cell membrane.
This can be achieved through chemical treatments that neutralize charges on the cell membrane or by exposing the bacteria to an electric field that creates microscopic pores in the cell membrane.
These methods yield chemically competent or electrocompetent bacteria, respectively. Following the transformation protocol, bacterial cells are plated onto an antibiotic-containing medium to inhibit the growth of the many host cells that were not transformed by the plasmid conferring antibiotic resistance. A technique called blue-white screening is then used for lacZ -encoding plasmid vectors such as pUC Blue colonies have a functional beta-galactosidase enzyme because the lacZ gene is uninterrupted, with no foreign DNA inserted into the polylinker site.
These colonies typically result from the digested, linearized plasmid religating to itself. However, white colonies lack a functional beta-galactosidase enzyme, indicating the insertion of foreign DNA within the polylinker site of the plasmid vector, thus disrupting the lacZ gene. Thus, white colonies resulting from this blue-white screening contain plasmids with an insert and can be further screened to characterize the foreign DNA. The bacterial process of conjugation see How Asexual Prokaryotes Achieve Genetic Diversity can also be manipulated for molecular cloning.
F plasmids , or fertility plasmids, are transferred between bacterial cells through the process of conjugation. Recombinant DNA can be transferred by conjugation when bacterial cells containing a recombinant F plasmid are mixed with compatible bacterial cells lacking the plasmid.
F plasmids encode a surface structure called an F pilus that facilitates contact between a cell containing an F plasmid and one without an F plasmid. On contact, a cytoplasmic bridge forms between the two cells and the F-plasmid-containing cell replicates its plasmid, transferring a copy of the recombinant F plasmid to the recipient cell.
Once it has received the recombinant F plasmid, the recipient cell can produce its own F pilus and facilitate transfer of the recombinant F plasmid to an additional cell.
The use of conjugation to transfer recombinant F plasmids to recipient cells is another effective way to introduce recombinant DNA molecules into host cells. Alternatively, bacteriophages can be used to introduce recombinant DNA into host bacterial cells through a manipulation of the transduction process see How Asexual Prokaryotes Achieve Genetic Diversity. In the laboratory, DNA fragments of interest can be engineered into phagemids , which are plasmids that have phage sequences that allow them to be packaged into bacteriophages.
Bacterial cells can then be infected with these bacteriophages so that the recombinant phagemids can be introduced into the bacterial cells. Molecular cloning may also be used to generate a genomic library. Having such a library allows a researcher to create large quantities of each fragment by growing the bacterial host for that fragment. These fragments can be used to determine the sequence of the DNA and the function of any genes present.
One method for generating a genomic library is to ligate individual restriction enzyme-digested genomic fragments into plasmid vectors cut with the same restriction enzyme Figure 5. After transformation into a bacterial host, each transformed bacterial cell takes up a single recombinant plasmid and grows into a colony of cells. All of the cells in this colony are identical clones and carry the same recombinant plasmid.
Figure 5. The generation of a genomic library facilitates the discovery of the genomic DNA fragment that contains a gene of interest. To construct a genomic library using larger fragments of genomic DNA, an E. Then, these recombinant phage DNA molecules can be packaged into phage particles and used to infect E.
During infection within each cell, each recombinant phage will make many copies of itself and lyse the E. Thus, each plaque from a phage library represents a unique recombinant phage containing a distinct genomic DNA fragment. Plaques can then be screened further to look for genes of interest. One advantage to producing a library using phages instead of plasmids is that a phage particle holds a much larger insert of foreign DNA compared with a plasmid vector, thus requiring a much smaller number of cultures to fully represent the entire genome of the original organism.
Figure 6. These recombinant phage DNA molecules are packaged into phage particles and allowed to infect a bacterial lawn. Each plaque represents a unique recombinant DNA molecule that can be further screened for genes of interest. Whereas all cells in a single organism will have the same genomic DNA, different tissues express different genes, producing different complements of mRNA.
This means that the introns, control sequences such as promoters, and DNA not destined to be translated into proteins are not represented in the library. The focus on translated sequences means that the library cannot be used to study the sequence and structure of the genome in its entirety.
The construction of a cDNA genomic library is shown in Figure 7. Figure 7. In contrast, plasmids utilized in the lab are usually artificial and designed to introduce foreign DNA into another cell. Minimally, lab-created plasmids have an origin of replication, selection marker, and cloning site.
The ease of modifying plasmids and the ability of plasmids to self-replicate within a cell make them attractive tools for the life scientist or bioengineer. The cloning method is ultimately chosen based on the plasmid you want to clone into. Regardless, once the cloning steps are complete, the vector containing the newly inserted gene is transformed into bacterial cells and selectively grown on antibiotic plates.
Importantly, because the bacteria from which plasmids are isolated grow quickly and make more of the plasmids as they grow, scientists can easily make large amounts of plasmid to manipulate and use in later work. Generally, scientists use plasmids to manipulate gene expression in target cells.
Characteristics such as flexibility, versatility, safety, and cost-effectiveness enable molecular biologists to broadly utilize plasmids across a wide range of applications.
Some common plasmid types include cloning plasmids, expression plasmids , gene knock-down plasmids, reporter plasmids, viral plasmids , and genome engineering plasmids. Addgene has compiled various educational resources to facilitate plasmid use in the lab. The guide also contains multiple protocols and troubleshooting tips to make plasmid usage as simple and straightforward as possible. If you have a question about a specific plasmid element that you would like answered or any topic suggestions for our Plasmids series , please let us know in the comments.
Topics: Plasmids , Plasmids. Add Comment. Addgene is a nonprofit plasmid repository. We archive and distribute high quality plasmids from your colleagues. Plasmids What is a plasmid? By Margo R. Figure 1: Map of a plasmid with its elements described below. Sharing science just got easier Subscribe to our blog.
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