Types of Cloning Vectors
Cloning is generally performed using Escherichia coli, and cloning vectors in E. coli include plasmids, bacteriophages, cosmids, and bacterial artificial chromosomes (BACs).
Some DNA, however, cannot be stably maintained in E. coli, for example very large DNA fragments. In this case, other organisms such as yeast may be used. Cloning vectors in yeast include yeast artificial chromosomes (YACs). The choice of classic cloning vectors depends on size of the insert and application [3] .
Plasmid
Plasmids are extra chromosomal double-stranded DNA sequences found in the cytoplasm of microbes and capable of replication using the host cell's replication machinery.
They are generally circular and stable genetic entity that can replicate itself autonomously, independent of the chromosomal DNA of the host organism.
entity 독립체
They are found widely in many bacteria, for example in E. coli, but may also be found in a few eukaryotes, for example in yeast such as Saccharomyces cerevisiae.
Plasmids usually carry at least one gene, and many of the genes that plasmids carry are beneficial to their host organisms.
Although they have separate genes from their hosts, they are not considered to be independent life [4] . Plasmids contain genes that enhance the survival of an organism, either by killing other organisms or by defending the host cell by producing toxins.
Multiple plasmids can coexist in the same cell, each with different functions. In other words, plasmids can only co-occur in a bacterium if they are compatible with each other.
coexist 공존하다
They are incompatible if they have the same reproduction strategy in the cell. An incompatible plasmid will be expelled from the bacterial cell [20] .
incompatible 공존할 수 없는
be expelled 파면을 당하다
Since plasmids are so small, they usually only contain a few genes that lend beneficial qualities to the organism. These genes are, however, non-essential for the survival of the organism.
The number of genes contained and the size of a plasmid varies from organism to organism. Some plasmids contain genes called transfer genes that facilitate the beginning of conjugation.
facilitate 가능하게 하다
Non-conjugative plasmids cannot start the conjugation process, and they can only be transferred through sexual conjugation with the help of conjugative plasmids.
Conjugation is the transfer of genetic material from one bacterial cell to another, either through direct contact or a bridge between the two cells [21] .
Plasmids can be eliminated from bacterial cells by a process called curing, which may take place spontaneously or by various curing treatments, such as acridine dyes, ultraviolet (UV) and ionizing radiation, thymine starvation and growth above optimal temperatures thatinhibit plasmid replication but do not affect bacterial chromosome replication and cell reproduction [17] .
eliminate 없애다
spontaneously 자연스럽게
They have three key points:The origin of replication, which is used to indicate where DNA replication is to begin; The selection marker gene, which is used to distinguish cells containing the plasmid from cells that do not contain it; and the cloning site, a site in the plasmid where the DNA is inserted [10] .
Based on function there are five main types of plasmids: (i) Fertility plasmids (also known as F-plasmids) contain transfer genes that allow genes to be transferred from one bacterium to another through conjugation.
(ii) Resistance plasmids or R plasmids, which contain genes that help a bacterial cell defend against environmental factors such as poisons or antibiotics. Some resistance plasmids can transfer themselves through conjugation.
(iii) Virulence Plasmids, inside a bacterium, turnthat bacterium into a pathogen. Example, E. coliand Salmonella enterica.
(iv) Degradative plasmids help the host bacterium to digest compounds that are not commonly found in nature, such as camphor, xylene, toluene, and salicylic acid. They are conjugative plasmids.
(v) Col plasmids contain genes that make bacteriocins (also known as colicins), which are proteins that kill other bacteria and thus defend the host bacterium. Some col plasmids are conjugative [6, 11 , 17] .
Use of plasmids asa vector
Plasmids are the standard cloning vectors and the ones most commonly used. Most general plasmids may be used to clone DNA insert of up to 15 kb in size.
One of the earliest commonly used cloning vectors is the pBR322 plasmid. Many plasmids have high copy number, for example pUC19 which has a copy number of 500-700 copies per cell and high copy number is useful as it produces greater yield of recombinant plasmid for subsequent manipulation.
subsequent 그 다음
manipulation 조작
However low-copy number plasmids may be preferably used in certain circumstances, for example, when the protein from the cloned gene is toxic to the cells [18] .
preferably선호되는
circumstances 상황
A plasmidis isolated from the bacterial cell at one site by restriction enzyme. The cleavage converts the circular plasmid DNA into a linear DNA molecule.
cleavage 분열
convert 전환시키다
Then the two open ends of linear plasmid are joined to the ends of the foreign DNA to be inserted with the help of enzyme DNA ligase. This regenerates a circular hybrid or chimeric plasmid, which is transferred to a bacterium wherein it replicates and perpetuates indefinitely [20] .
regenerate 재생되다
perpetuate 영구화하다
indefinitely 무기한으로
Significance of plasmids dramatically increased with the advent of recombinant DNA technology as they became the first cloning vectors, and even today they are the most widely used cloning vectors especially in gene cloning in bacteria.
Significance 중요성
advent 출현
They enjoy this status because they have very useful properties that include: small size, which makes the plasmid easy to isolate and manipulate; independent origin of replication, which allows plasmid replication in the cell to proceed independently from direct chromosomal control; multiple copy number, which makes them to be present in the cell in several copies so that amplification of the plasmid DNA becomes easy; presence of selectable markers such as antibiotic resistance genes, which make detection and selection of plasmid-containing clones easier; and cloning site (MCS), also called a polylinker site, which is a stretch of DNA that consists of multiple target sequences for specific restriction enzymes.
properties 성질
manipulate 조종하다
amplification 증폭
stretch 늘이다
The plasmid vectors also possess promoter sequences near the MCS for efficient expression of gene of interest [1] .
sequence 배열 순서를 밝히다
Plasmids often confer antibiotic resistance to the bacteria, so only bacteria containing the vector will survive treatment with antibiotics. Antibiotic resistance is often used as marker, an example is the beta-lactamase gene which confers resistance to the penicillin group of beta-lactam antibiotics like ampicillin [7].
One of the first widely used E. coli cloning vectors is pBR322 DNA.It is a double-stranded circle molecule with cloning limit of 0.1-10 kb. It was created in 1977 by Z. Bolivar and F. Rodriguez at the University of California. The p stands for "plasmid" and BR for "Bolivar" and "Rodriguez.". pBR322 DNA is 4361 base pairs in length and has two antibiotic resistance genes - the gene bla encoding the ampicillin resistance (AmpR) protein, and the gene tetA encoding the tetracycline resistance (TetR) protein. It contains the origin of replication of pMB1, and the rop gene, which encodes a restrictor of plasmid copy number. The plasmid has unique restriction sites for more than forty restriction enzymes [20] . The circular sequence is numbered such that 0 is the middle of the unique EcoRI site and the count increases through the TetR gene. The AmpR gene is penicillin beta-lactamase. Promoters P1 and P3 are for the beta-lactamase gene. P3 is the natural promoter, and P1 is artificially created by the ligation of two different DNA fragments to create pBR322. P2 is in the same region as P1, but it is on the opposite strand and initiates transcription in the direction of the tetracycline resistance gene [2] .