Multiple cloning site

A pUC19 cloning vector showing the multiple cloning site sequence with restriction enzyme sites

A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites—a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. The purpose of an MCS in a plasmid is to allow a piece of DNA to be inserted into that region.

An MCS is found in a variety of vectors, including cloning vectors to increase the number of copies of target DNA, and in expression vectors to create a protein product. In expression vectors, the MCS is located downstream of the promoter.

Creating a multiple cloning site

In some instances, a vector may not contain an MCS. Rather, an MCS can be added to a vector. The first step is designing complementary oligonucleotide sequences that contain restriction enzyme sites along with additional bases on the end that are complementary to the vector after digesting. Then the oligonucleotide sequences can be annealed and ligated into the digested and purified vector. The digested vector is cut with a restriction enzyme that complements the oligonucleotide insert overhangs. After ligation, transform the vector into bacteria and verify the insert by sequencing. This method can also be used to add new restriction sites to a multiple cloning site.

A diagram showing the process of inserting a multiple cloning site into a plasmid vector

Uses

Multiple cloning sites are a feature that allows for the insertion of foreign DNA without disrupting the rest of the plasmid which makes it extremely useful in biotechnology, bioengineering, and molecular genetics. MCS can aid in making transgenic organisms, more commonly known as a genetically modified organism (GMO) using genetic engineering. To take advantage of the MCS in genetic engineering, a gene of interest has to be added to the vector during production when the MCS is cut open. After the MCS is made and ligated it will include the gene of interest and can be amplified to increase gene copy number in a bacterium-host. After the bacterium replicates, the gene of interest can be extracted out of the bacterium. In some instances, an expression vector can be used to create a protein product. After the products are isolated, they have a wide variety of uses such as the production of insulin, the creation of vaccines, production of antibiotics, and creation of gene therapies.

Structural features in vector types

MCSs have distinct structural features depending on the type of vector in which they are used.

In cloning vectors, MCSs are typically places within a selection marker, such as the lacZα gene in pUC vectors. This configuration allows for efficient screening for recombinant plasmids because the insertion of foreign DNA into the MCS inactivates the marker gene, allowing for blue-white screening or other selection methods.

In expression vectors, MCSs are used between a promoter and a terminator in order to regulate gene expression. The upstream promoter can be either constitutive or inducible and respond to specific chemical inducers, while the downstream terminator facilitates proper termination of transcription and enhances plasmid stability.

In reporter vectors, an MCS is typically placed near a reporter gene, for example, a fluorescent protein (GFP), luciferase, or lacZ. This allows promoter sequences to be added to the MCS to facilitate the study of promoter activity and gene regulation by monitoring reporter gene expression.

Historical Background

Early Developments of Cloning Vectors

Stanley N. Cohen and Herbert W. Boyer conducted experiments in 1973 demonstrating that genes from a different species could be inserted into bacterial cells and expressed. They employed the plasmid pSC101, a naturally occurring plasmid from the bacterial species Salmonella panama, which they altered to have a single EcoRI restriction site and a tetracycline resistance gene. The pSC101 plasmid did not have a formal multiple cloning site, but its design highlighted the utility of restriction sites for gene insertion, paving the way for the later creation of dedicated MCS regions in vectors.

Introduction of pBR322 and Enhanced Cloning Sites

From previous research, in 1977, scientists Francisco Bolivar and Raymond L. Rodriguez, built the pBR322 plasmid. While not being officially a multiple cloning site, this plasmid was one of the first vectors to have more than one unique restriction site. The sites were inserted into a strategic location in its sequence, such as in antibiotic resistance genes, where they could be used for insertional inactivation to identify recombinant clones. This introduced greater flexibility in the insertion of foreign DNA fragments and marked a considerable progression toward having designated MCS regions in future vectors, such as the pUC sites.

Construction of pUC Vectors and Formal Definition of MCS

The pUC vector series was built in 1982 by Jeffrey Vieira and Joachim Messing, starting from M13mp7. The vectors had a particular region defined as having numerous independent restriction sites and formally defining the concept of the MCS. The pUC vectors made the sequencing and cloning, significantly enhancing the molecular cloning procedure.

Modern design and optimization of the MCS

Present advancements in MCS design have made cloning more efficient, flexible, and easy to perform, which makes them frequently used in molecular biology.

MCS design

Restriction site placement and strategic selection in MCSs optimize flexibility and compatibility and reduce potential cloning issues. Also facilitating greater workability and versatility for accommodating a vast range of experiments and applications. In an MCS, the occurrence of multiple unique restriction sites in proximity allow for less constraints on enzyme selection. Such a design enables enzymatic cleavage at specific positions, enabling specific MCS changes for various applications. Widely used plasmids such as pUC19 and other pUC plasmids have purposefully placed restriction sites to facilitate effective cloning, contributing to the flexibility of MCS in molecular use.

Removal of undesirable restriction sites

Improvements in bioinformatics and molecular techniques enable identification and elimination of undesirable restriction sites, thus streamlining the process of cloning. Bioinformatic tools assist in screening and identification of unwanted restriction sites in the MCS of concern or vector backbone. They have the potential to cause significant variation in protein expression depending on their position, and some impose very sharp restriction of expression of a gene of interest if located in the MCS. By hitting and removing these problematic sites, researchers are able to create MCSs to obtain uniform and effective protein expression.

Modularity and flexibility

Modern MCSs are designed with modularity in mind, making it easier to integrate and swap out to genetic elements, which is particularly beneficial in the field of synthetic biology. Standardized flanking genetic sequences in MCS construction allow easy replacement of the genetic elements. Modular design enables rapid assembly and customization of vectors for specific research needs. The MoClo system allows for efficient assembly of DNA fragments into multigene constructs with a modular design. The system allows ease of coupling DNA fragments (unwanted sequences-free) to make the process size-effective and efficient. For example, the MoClo system allows for efficient coupling of two genes, e.g., a promoter sequence and a coding sequence, each from different MSCs, into a single multigene construct without incorporating unwanted sequences.

Compatibility with advanced cloning technologies

Contemporary MCSs are designed to be compatible with advanced cloning technologies, enhancing the accuracy and efficiency of the genetic manipulations. Contemporary MCS designs facilitate such technologies as Gibson Assembly and Golden Gate cloning that have advantages over the traditional restriction enzyme-based protocols, such as parallel assembly of multiple DNA pieces. Such compatibility enhances the efficiency and usefulness of the MCS region. This compatibility is essential in genetic engineering and synthetic biology, in which the effective and precise assembly of various genetic components is essential to construct intricate genetic circuits or metabolic pathways.

Optimizing sequence context

Strict attention to sequence context near MCSs ensures effective cloning and proper gene expression. Elimination of secondary structures that are possible between DNA components in the MCS (such as promoters and open reading frames) prevents interference with restriction enzyme activity and optimizes MCS activity. Secondary structures within the 5' untranslated region, for instance, can prevent ribosome binding, reducing translation efficiency. In addition, preventing the placement of stop codons and preventing the interruption of reading frames eliminates undesirable mutations or translational errors, such as premature stop codons that may truncate the protein product, further optimizing MCS reliability in vectors.

Challenges and limitations

Despite optimizations and advancements, certain challenges persist in MCS design, necessitating ongoing research and innovation. One of these issues is the occurrence of internal restriction sites in genes or vectors, which can interfere with restriction enzyme activity and cloning efficiency. Furthermore, the structural environment of MCSs can confer unintended regulatory properties. For instance, MCSs inserted too far away from promoter regions can result in secondary structures formation, interfering with expression of a gene in an MCS. In addition, sequence context variability (e.g., differences in proximity to promoters or proximal regulatory elements) can lead to uneven gene expression. This variability arises as a result of variations in the surrounding sequences, which can interfere with transcription efficiency, mRNA stability, or initiation of translation. In order to minimize such variability, innovations beyond are needed to engineer standardized MCS designs that yield uniform performance in diverse genetic constructs.

Example

One bacterial plasmid used in genetic engineering as a plasmid cloning vector is pUC18. Its polylinker region is composed of several restriction enzyme recognition sites, that have been engineered into a single cluster (the polylinker). It has restriction sites for various restriction enzymes, including EcoRI, BamHI, and PstI. Another vector used in genetic engineering is pUC19, which is similar to pUC18, but its polylinker region is reversed. E.coli is also commonly used as the bacterial host because of the availability, quick growth rate, and versatility. An example of a plasmid cloning vector which modifies the inserted protein is pFUSE-Fc plasmid.

In order to genetically engineer insulin, the first step is to cut the MCS in the plasmid being used. Once the MCS is cut, the gene for human insulin can be added making the plasmid genetically modified. After that, the genetically modified plasmid is put into the bacterial host and allowed to divide. To make the large supply that is demanded, the host cells are put into a large fermentation tank that is an optimal environment for the host. The process is finished by filtering out the insulin from the host. Purification can then take place so the insulin can be packaged and distributed to individuals with diabetes.