DNA Transfer and Genetic Engineering

Micro-injection is a method used to Inject a recombinant DNA into the nucleus of an animal cell. This is the only way to introduce alien DNA into host cells. 

Alternative selectable markers have been developed which differentiate recombinants from the non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. In this, a recombinant DNA is inserted within the coding sequence of an enzyme, ( -galactosidase. This results into inactivation of the enzyme, which is referred i to as insertional inactivation. The presence of a chromogenic j substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertional inactivation of the -galactosidase and the colonies do not produce any colour, these are identified as recombinant colonies.

Alternative selectable markers have been developed which differentiate recombinants from the non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. In this, a recombinant DNA is inserted within the coding sequence of an enzyme, ( -galactosidase. This results into inactivation of the enzyme, which is referred i to as insertional inactivation. The presence of a chromogenic j substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertional inactivation of the -galactosidase and the colonies do not produce any colour, these are identified as recombinant colonies.

The product produced by the bacteria that digests the antibiotic (like ampicillin) is a protein (an enzyme like beta-lactamase). In the context of recombinant DNA technology, this is a recombinant protein product.

In order to force a bacteria to take up the plasmid , the bacterial cells must to be made competent. This is done by treating them with a specific concentration of divalent cations such as calcium which increases the efficiency with which the DNA molecule enters into the bacteria through pores in its cell wall. Recombinant DNA then forced into such cells by incubating the cells with recombinant DNA on ice followed by placing them immediately at heat shock and then again into ice. This enables the bacteria to take up the recombinant DNA. 

Selectable markers have been developed which differentiate recombinants from non- recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. In this, a recombinant DNA is inserted within the coding sequence of an enzyme B-galactosidase. This results into an  insertional inactivation. The presence of chromogenic substrate gives blue coloured colonies if the plasmid in bacteria does not have an insert. Presence of insert results into insertional inactivation of a^-galactosidase enzyme and the colonies do not produce any colour, these are identified as recombinant colonies. 

The blue-white screening method uses a modified plasmid vector (like pUC19) in E. coli to detect recombinants based on the inactivation of the lacZ gene, which produces a color change.

• Recombinant DNA molecules are sometimes called chimeric DNA, because they can be made of material from two different species, like the mythical chimera.
• A chimera is essentially a single organism that is made up of cells from two or more individuals—that is, it contains two sets of DNA, with the code to make two separate organisms.
•  R-DNA technology uses palindromic sequences and leads to the production of sticky and blunt ends.

Till today, the most important in genetic engineering of plants has been the Ti plasmid of soil bacterium, Agrobacterium tumefaciens. E.Coli  has been extensively used as "work horse" for genetic engineering e.g., production of humulin, somatotropin. E. coli is one of the most thoroughly studied of all living things. It is a favorite organism for genetic engineering as cultures of it can be made to produce unlimited quantities of the product of an introduced gene. Several important drugs (insulin, for example) are now manufactured in E. coli. Thus, option B is correct. Nitrosomonas, Klebsiella, Nitrobacter, Azotobacter, Rhizobium and Diplococcus are not used in genetic engineering.

Genetic engineering is a branch of science that uses various biotechnological techniques to manipulate an organism's genome to  create species with desired traits. However, the process involves isolation, transfer and insertion of the new genetic material into host genomes and storage of genomes in organisms and selective markers for an indefinite period. Experiments have shown that certain bacteria has the ability to naturally take in and express foreign DNA.  It has been found that of these,  E.coli  has the ability to transfer DNA laterally to and from other species while Agrobacterium  can transfer genes to plants and fungi by causing crown gall disease.

Escherichia coli and Agrobacterium tumefaciens are the most widely used bacteria in genetic engineering for cloning and plant transformation, respectively.

Option 'D' is correct.

Genetic engineering is a branch of science which uses a set of biotechnology techniques and knowledge of cytology and genetics to  bring modifications to an organism's genome.   For such research,  scientists need to store and modify the genes and genetic information from wide range of organisms into organisms that can be easy to transform.  Bacteria more commonly Escherichia coli is used to transform and create the genetically modified bacteria as this can be transformed and stored easily for indefinite periods at -80 C and replicate the modified DNA in short time period.

Transformation is the process by which a bacterial cell takes up naked DNA from its environment.

When two different mutant strains of T4 bacteriophage (R-IIx and R-IIy) infect the same E. coli host cell, they can undergo complementation or recombination because they have mutations in different cistrons (genes). This allows them to produce functional proteins and successfully lyse the bacterium.

In blue-white screening, the insertion of foreign DNA into the lacZ gene (which encodes beta-galactosidase) causes insertional inactivation, preventing the production of the enzyme and resulting in white colonies.

Reporter gene technology links a putative promoter or enhancer region to the coding region of an easily assayable reporter gene, allowing rapid and sensitive measurement of changes in transcription levels.

Genetic engineering, also known as recombinant DNA technology, means altering the genes in a living organism to produce a genetically modified organism (GMO) with a new genotype. Various kinds of genetic modification are possible: inserting a foreign gene from one species into another, forming a transgenic organism; altering an existing gene so that its product is changed; or changing gene expression so that it is translated more often or not at all.