Cloning Vectors in Genetic Engineering

 Hello friends, in today's article, we see different types of cloning vectors in Genetic engineering.

so let's see one by one

Cloning Vector in Genetic Engineering:-

first, let's understand what is the meaning of cloning.

Cloning:-

- Cloning is the process of generating a genetically identical copy of a cell or an organism.

- Cloning happens often in nature for example when a cell replicates itself asexually without any genetic alternation or recombination.

- In Bacteria create genetically identical duplicates of themselves using binary fission or budding.

- In humans, all the cells that undergo mitosis division, such as skin cells and cells lining the gastrointestinal tract, are clones exceptions is eggs and sperm ( gametes)

Cloning used in Genetic engineering:-

- Molecular cloning refers to the isolation of a DNA sequence from any species (gene) and its insertion into a vector for propagation without changing the original DNA Sequence.

- Molecular clones can be used to generate many copies of the DNA for analysis of the gene sequence and express the resulting protein for the study or utilization of the function of the protein.

- the clones can also be manipulated and mutated invitro to alter the expression and function of the protein.

Gene-cloning Steps:-

the basis cloning workflow includes four steps

1) Isolation of Target DNA 

2) Ligation ( insert DNA fragment on appropriate cloning vector, creating recombinant molecules e.g. plasmids)

3) Transformation of recombinant plasmids into bacteria for propagation.

4) Screening of Hosts containing the intended recombinant plasmids.

Cloning Vectors:-

- A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.

- The cloning vector may be DNA was taken from a virus, the cell of a higher organism or it may be the plasmid of the bacterium.

- It is a DNA Molecules that have the ability to replicate in an appropriate host cell and into which the DNA fragment to be cloned is integrated for cloning.

- Therefore, a vector must an origin of DNA replication that functions in the host cell.

- Any extra-chromosomal small genome, e.g. Plasmid, phage, or virus, may be used as a vector.

- Herbert Boyer, Keiichi Itakura, and Arthur Riggs recognized a general cloning vector with unique restriction sites for cloning in foreign DNA and the expression of antibiotic resistance genes for the selection of transformed bacteria.

- In 1977, they described the first vector for cloning purposes, pBR 322 a plasmid

- this vector was small, 4kb in size, and had two antibiotic resistance genes for selection.

History of Vectors:-

- In 1961, the first genetic recombination demonstrated happened.

- In 1967, the isolation of the first DNA ligases.

- In 1968, the isolation of the first striction factor that could selectively cut bacteriophage DNA

- in 1970, the isolation of restriction enzymes ( selectively cut DNA)

- In 1971, the restriction enzymes mapping of the simian virus SV40

- In 1972, Assembly of the first recombination DNA and transformation of the first E.coli.

- In 1974, NEB opens for business

- In 1975, Launch a RABASE ( restriction enzyme database)

- in 196-77, Introduction of Maxum-gilbert and sanger sequencing

- In 1977, a Report of the first cloning vector pBR 322

- in 1978, the Noble prize was awarded to smith, Arber, and Nathans for the discovery of restriction enzymes.

Properties of Vectors:-

- It should be able to replicate autonomously, when the objective of cloning is to obtain a large number of copies of the DNA insert, the vector replication must be under relaxed control so that it can generate multiple copies f itself in a single host cell.

- A vector should be ideally less than 10kb in size, because, large DNA molecules are broken during the purification procedure. In addition, large vectors present difficulties during various manipulations required for gene cloning.

- the vector should be easy to isolate and purify.

- It should be easily introduced into the host cells i.e. transformation of the host cell with the vector should be easy.

- The vector should have suitable marker genes that allow easy detection and selection of the transformed host cells.

- When the objective is gene transfer, it should have the ability to integrate either itself or the DNA insert it and carries into the genome of the host cell.

- The cells transformed with such vector molecules that contain the DNA insert ( recombinant DNA ) should be identifiable or selectable from those transformed by the vector molecules only.

- A vector should contain unique target sizes for as many restriction enzymes as possible into which the DNA insert can be integrated without disrupting an essential function.

- When expression of the DNA insert is derived the vector should contain at least suitable control elements e.g. promoter, operator, and ribosome binding sites, several other features may also be important

Selections of Vector:-

- Insert size ( depends on how much they can carry)

- For projects in which it is derived that's a particular piece of DNA be cloned on consideration is the size of the insert DNA.

- Copy number of vector

- Incompatibility of vector

- Selectable Marker

- Cloning sites may present

- Specialized Plasmid functions.

Properties of Host:-

- Host should be easy to transform.

- Host should be free from elements that interfere with the replication of recombinant DNA.

- Host should have lack active restriction enzyme, E.coli, K2 , strain HB101

- Host Should not have methylases since these enzymes would methylate the replicated recombinant DNA as a result, would become Resistant to useful restriction enzymes 

-and Should be deficient in normal recombination function so that the DNA insert is not altered by recombination events.

E.coli used as Host for cloning:-

-  E.coli supports several types of Vector some natural, some constructed

- Plasmid, bacteriophage, phasmids, shuttle vectors, cosmids, Artificial chromosome, Phagemids

so this is all about the Cloning vector in genetic engineering.

Read More:-

1) Nucleic Acids MCQ's