Introduction
After a gene or part of a gene has been isolated using PCR or restriction digestion, the next step in the plasmid design process is to insert the DNA into the plasmid vector being cloned. In genetic engineering, a vector is an agent, typically a plasmid, that is inserted into a cell for the purposes of genetic propagation.
As discussed in my initial sections, cloning vectors often originate from bacterial plasmids, which are circular DNA molecules ranging from 2,000 to 100,000 base pairs (bp) in length. However, most cloning plasmids are within the range of 2,000 to 10,000 bp. Bacteria can naturally harbor multiple copies of a specific plasmid or possess single copies of various plasmids. Plasmids have the ability to replicate independently of the host DNA and commonly carry one or more genes. These genes often encode factors that aid the survival of bacteria. For instance, plasmids may carry genes providing resistance to antibiotics like ampicillin. Moreover, plasmids can be transferred between different bacteria. These attributes have led to both remarkable applications in cloning, enabling numerous molecular biology techniques, and the emergence of pathogenic bacteria resistant to multiple antibiotics.
Favorable Attributes of a Vector
Plasmids possess inherent qualities that make them suitable as cloning vectors, and additional desirable attributes have been incorporated through genetic engineering. There is a wide range of commercially available vectors designed for various applications. A cloning vector differs from an expression vector or a tagging vector in its purpose. The fundamental characteristics of an effective vector include:
Self-replication: Plasmids contain an origin of replication that enables them to replicate autonomously within the host cell. Since most cloning vectors utilize a bacterial origin of replication, they can be replicated by the enzymes already present in the host bacteria.
Size: Bacterial vectors are typically small, ranging from 2,000 to 10,000 base pairs (2–10 kilobases or kb), facilitating their manipulation.
Copy number: Each plasmid exists at specific levels within its host bacterial strain. A high-copy-number plasmid may have numerous copies in each bacterium, while a low-copy-number plasmid might have only one or two copies per cell. Cloning vectors derived from specific plasmids exhibit the same copy number range as the original plasmid. Most commonly used vectors have a high copy number.
Multiple cloning site (MCS): Vectors are engineered to incorporate an MCS, which is a collection of restriction sites. This feature simplifies the insertion of foreign DNA into the plasmid. An MCS typically contains 20 or more unique enzyme sites, with each site being exclusive to both the MCS and the plasmid. This ensures that the corresponding restriction enzyme will only cleave the plasmid at its specific site within the MCS.
Selectable markers: Plasmids can carry one or more resistance genes for antibiotics. If the transformation is successful (i.e., the plasmid enters and replicates within the host cell), the host cell will be able to grow in the presence of the antibiotic. Therefore, antibiotics serve as markers to select for positive transformants.
Screening: In the process of transforming bacteria with a ligation reaction, not all the vectors will necessarily contain the desired DNA fragment. To facilitate the identification of cells that carry an insert, vectors often incorporate reporter genes that differentiate them from cells lacking inserts.
Positive selection: Some newer plasmid vectors employ positive selection, wherein the inserted DNA disrupts a gene that would otherwise be lethal to the bacteria. If foreign DNA fails to insert into the MCS, the lethal gene is expressed, resulting in the death of transformed cells. However, successful insertion of foreign DNA prevents the expression of the lethal gene, allowing the transformed bacteria to survive and proliferate. Positive selection eliminates the need for reporter genes, as only cells transformed with vectors containing an insert will survive.
Control mechanism: Most vectors incorporate a control mechanism for regulating the transcription of the antibiotic resistance gene or other engineered genes. One well-known control mechanism is the lac operon, which consists of a group of genes. In the absence of lactose, the lac repressor protein binds to the lac operon, preventing gene transcription. When lactose is present, it binds to the lac repressor protein, causing it to dissociate from the operon. This allows RNA polymerase to bind, facilitating gene transcription. Lactose serves as an inducer of the lac operon. Genes from the lac operon have been incorporated into many cloning vectors.
Size of insert: Plasmid vectors have limitations on the size of inserts they can accommodate, typically smaller than the size of the vector itself. Other vectors have been developed to handle larger target DNA fragments.
DNA Ligation
Ligation refers to the process of combining an insert DNA with a vector DNA to achieve molecular cloning. This is typically aided by an enzyme known as DNA ligase. DNA ligase facilitates the formation of a phosphodiester bond between the 3' hydroxyl group of one DNA fragment and the 5' phosphate group of another DNA fragment.
In the previous section on restriction digestion, the process of cutting DNA strands using an enzyme was explained; the ligation process works in reverse. Just as with restriction digestion, there are two methods of ligating DNA: one involves using DNA with “sticky ends,” and the other involves using DNA with “blunt ends.” DNA with sticky ends contains unpaired bases at its ends, creating an overhang without complementary bases on the other strand. In contrast, DNA with blunt ends lacks these overhangs. When a DNA fragment is generated through PCR, it typically produces sticky ends with a single adenosine (A) overhang. On the other hand, when a DNA fragment is produced by cutting a DNA segment with a restriction enzyme, it can result in either sticky ends or blunt ends, depending on the specific restriction enzyme used.
In the case of ligation with sticky ends, to prepare a cloning vector for ligation with insert DNA, the vector is first cut with a restriction enzyme within the MCS (Multiple Cloning Site), creating an opening to accept the inserted DNA. If the insert DNA possesses sticky ends, which are overhangs on the ends of the DNA strands, the vector should be cut with the same enzyme. This process generates sticky ends on the vector that are complementary to the ends of the insert DNA. For instance, if the insert DNA has been cleaved at both ends using Bgl II, then the vector would also be cleaved with Bgl II. Ensuring complementary sticky ends enhances the efficiency of ligation, whereas mismatches in the sequences make the insertion of DNA less likely to occur. Since the sticky ends on both the vector and the insert DNA are complementary, they will form base pairs when they encounter each other during the ligation process.
Sticky-end ligation offers the advantage of enabling directional cloning. If it is desired to have the insert DNA in a specific orientation (e.g., A ==> B direction in the vector but not B ==> A direction), both the insert and vector can be digested with different restriction enzymes, resulting in asymmetric ends. This is particularly crucial when the DNA insert is a cDNA intended for expression in the transformed cell. By doing so, only the complementary ends will successfully ligate, ensuring that the insert maintains a single orientation in the ligation products.
Blunt-end ligation involves the joining of DNA fragments where there are no overhangs or unpaired bases. This method offers an advantage over sticky-end ligation as all DNA ends are compatible with each other, eliminating the need to cut the vector and insert with specific restriction enzymes to generate complementary overhangs. Blunt-end ligation vectors contain a ligation site in the MCS that accepts blunt-ended DNA fragments. Despite lacking overhangs, these vectors still retain the MCS, which is valuable for further manipulation of the inserted DNA using restriction enzyme sites.
Ligation Gone Wrong
Ligation is a very inefficient process; from millions of vectors and inserts, only 1–100 are expected to ligate together as desired and lead to growth of colonies from the desired transformants. The desired outcome is the successful ligation of a single insert into the vector. With blunt-end inserts and vectors where directionality is lacking, the orientation of the insert can be either forward or reverse, resulting in a roughly equal distribution of both orientations in the ligation products. However, there can be several possible undesirable products from a ligation:
• Self-ligation of the vector — a self-ligated vector, without any DNA inserted in the MCS, should have an intact lethal gene. The product of the lethal gene should kill any bacteria transformed by these vector-only constructs.
• Ligation of a vector with primers or other short DNA fragments — even if these fragments are a small proportion of the total fragments available for ligation, small fragments ligate more easily than larger fragments.
• Ligation of a vector with multiple inserts — in the case where inserts all have the same blunt ends, they can ligate to each other (a process known as concatenation) and also ligate to the vector, giving a product with multiple inserts in a row. The number of these products can be reduced by controlling the molar ratio of insert to vector. If the ratio is high (that is, if there are many more insert molecules than vector), then it is more probable that inserts will ligate to each other rather than to the vector. So the molar ratio can be an important factor in setting up ligation reactions.
• Self-ligation of insert — it is possible for the insert molecules to self-ligate, forming closed circles. Since these molecules do not have any of the vector genes, they will not be able to replicate to form colonies, so they are not of consequence.
Conclusion
Ligation plays a pivotal role in DNA cloning and gene manipulation through the construction of genetic constructs. Careful experimental design, proper controls, and optimization are essential for successful ligation. Advances in ligation techniques have greatly facilitated genetic engineering, enabling the development of recombinant DNA molecules that have revolutionized research, biotechnology, and medicine. Further research in ligation methods will continue to drive advancements in molecular biology and expand the possibilities of genetic manipulation.
References
Shuman S (June 2009). “DNA ligases: progress and prospects”. The Journal of Biological Chemistry. 284 (26): 17365–17369.
Sambrook J, Russell D. “Chapter 1: Plasmids and Their Usefulness in Molecular Cloning”. Molecular Cloning — A Laboratory Manual. Vol. 1 (3rd ed.). pp. 1.20–1.21. ISBN 978–0–87969–577–4.
Bio-Rad Explorer Team, “Cloning and Sequencing Explorer Series.” Bio-Rad Laboratories, Catalog # 1665000EDU.
“Plasmids 101: Addgene.” Team:Ku Istanbul, 2020.igem.org/Team:KU_ISTANBUL.
Rye, Connie. “10.1 Cloning and Genetic Engineering.” Concepts of Biology 1st Canadian Edition, 14 May 2015, opentextbc.ca/biology/chapter/10–1-cloning-and-genetic-engineering
