Biotechnology : Principles And Processes

In baking industry yeast is used with flour; yeast undergoes fermentation thereby releasing ethanol and CO_2. The production of this gas causes rise in dough, making it soft and spongy.

The enzymes belonging to the group nucleases are known to cut nucleotide sequences.

Endonuclease makes cut within the DNA at specific site, whereas the exonucleases cut the nucleotide sequence present at the end.

Hind II is the restriction endonuclease. DNA ligase is involved in joining of two DNA fragments.

•Conjugation. It is the phenomenon of transfer of the genetic material directly from one bacterial cell to the other bacterial cell via a physical bridge called the conjugation tube.

•Transformation. It involves direct uptake of genetic material through the cell membrane.

•Translation. In this process the RNA encodes for protein. Related with gene expression.

•As electric field is applied, DNA fragment arrange themselves according to the size.

•These DNA fragments are then stained with Ethidium bromide and exposed to UV light.

•The DNA band becomes visible and appears as orange coloured bands.

•Restriction enzymes are known to cut nucleotide bases, also called molecular scissors.

•Divided into two types:

a) Endonuclease: these are known to bind with specific sequence present within the DNA, then cut the pallindromic sites.

b) Exonuclease: these enzymes are to remove nucleotides from the ends of DNA.

•Restriction enzymes act as molecular scissors and cut the DNA into fragment.

•DNA ligase is used to construct rDNA, by joining fragment of DNA [gene of interest] with the DNA of genetic material of the vector.

Principle involved in gel electrophoresis:

When electric field is applied the DNA (negatively charged) start to move towards the anode. This helps the lighter or smaller fragments to travel at faster rate, hence they resolve according to the size.

Smallest fragment would be nearer to the anode plate side.

Ori is the most important site in a vector due to following reasons:

a) It controls the copy number.

b) It’s the site for the insertion of gene of interest.

c) Ori site initiates replication of DNA.

If DNA treated with Deoxyribonuclease it would result in it’s digestion. Rather than isolation, denaturation would happen.

In PCR during extention phase, taq polymerase is used to add nucleotide at a very high temperature of 72⁰ C. Thermostable Taq polymerase remains stable and active even at such a high temperature.

•Antibiotic resistance genes act as selectable markers.

•In the host cell DNA, when gene of interest gets incorporated, the host cell is called transformant.

•Antibiotic resistance gene like PVU [Ampicillin resistance gene] is used to distinguish between transformants and non-transformants.

•The host is rubbed by diavalent cation like Ca²⁺, this results in the increase in diameter of pores of bacterial host cell wall.

•The host and rDNA are than kept on ice, then heated at 42⁰ C and again kept on ice.

•This treatment makes the cell wall competent and allow to uptake foreign DNA.

•DNA ligase joins the two DNA fragment by forming phosphodiester bonds.

•Also known as ‘Genetic gums’.

•Agrobacterium tumificiean is genetically engineered to make it act as a vector.

•The pathogenic gene of Ti plasmid has been disarmed, which is known to induce tumour formation.

Taq Polymerase is a thermostable DNA polymerase which can extent the primers by adding nucleotide even at a high temperature of 72⁰C.

•Introns are the long sequences of RNA found in eukaryotic organisms.

•These sequences do not code for any proteins.

•As bacteria lacks RNA splicing machinery, the gene expression is blocked in prokaryotes.

•These include open system bioreactors which are opened at regular intervals and new media can be continuously added.

•The cell multiplies at faster rate, and the previous culture media is taken out while fresh media is added.

In 1985 Kary Mullis developed the technique for the amplification of DNA in number called PCR.

•Restriction enzymes are obtained from bacteria.

•Restriction enzymes are the defensive mechanism present in the bacteria.

•Copy number decides the times to which the rDNA can be multiplied.

•Hence, it can be said that more are the copy number higher amount of the recombinant protein is produced.

•No, exonuclease removes nucleotide from the end of the DNA strands.

•If DNA is treated with exonuclease, no overhanging sticky ends would be produced. Hence rDNA could not be constructed.

H refers to the organisms genus from which enzyme has been isolated. H refers to haemophilus.

d refers to the strain type. III refers to the sequence in which the restriction enzyme is isolated.

It would result in formation of multiple fragments, if a restriction enzyme would have more than one site of action.

Competent cell refers to the capability of the cell wall of bacteria to uptake hydrophilic DNA fragments through cell membrane when treated with diavalent ions.

•Proteases are the enzymes which digest the protein present inside the cell.

•Proteases ensure the isolation of DNA without its contamination with proteins.

•Moreover, protein causes interference during downstream processing.

If the denaturation step is missed, DNA strands would not separate from each other hence primer could not be added to the ends.

•It would disrupt the whole process of amplification.

Hepitisis-B vaccine.

•No, in absence of water biomolecules become dysfunctional.

•Water is involved in H-bonding hence it plays an important role in the maintenance of the structure of DNA and protein.

•If H-bond weakens or collapses, it results in denaturation of biomolecules.

•Ti plasmid of Agrobacterium tumificiens possesses the capacity to induce tumour formation.

•It has been genetically disarmed, by deleting the gene responsible for the tumour formation.

•Due to presence of a plasmid with ori site with high copy no. It is used as a cloning vector.

•Gene cloning involves the insertion of gene of interest within the DNA of a vector.

•These vectors are used to introduce the gene of interest inside the host cell.

•These host cell are known as transformants.

•Both approaches are correct.

•Wine maker uses yeast to produce ethanol (natural phenomenon).

•Whereas a molecular Biologist uses vector with high copy number to produce large amounts of antigens.

•It would not affect the constructed recombinant DNA.

•Recombinanat DNA being circular has no free ends, hence the endonuclease would not recognise the specific pallendromic nucleotide sequences and will not cut at specific positions.

If restriction enzymes don’t cut the DNA at specific positions, it would not produce sticky ends and construction of recombinant DNA would not be possible.

•When endonuclease makes cut on the specific restriction site of the circular DNA of plasmid, the circular DNA changes into linear DNA.

•But, when endonuclease makes cut within the linear DNA it would result in formation of two linear DNA fragments.

DNA is made visible by staining it with Ethidium bromide, which on exposure with UV rays appears to be orange colored bands.

•Absence of selectable marker would not affect the construction of recombinant DNA.

•But in absence of selectable marker, it would become difficult to distinguish between transformant and non transformant cell.

After staining the agarose gel with Ethidium bromide,if no DNA bands are observed it may occur due to following reasons:

•May be the isolated DNA has been degraded during the isolation process.

•May be the DNA is not exposed to UV radiations after staining it with Ethidium bromide .

•CaCl₂ is known to make bacterial cell wall competent.

•When Ca²⁺ ions are rubbed against the cell wall it results in enlargement of the diameter of the pores and allows the uptake of foreign DNA fragment.

In absence of anti biotic mixture of both transformants and non transformants will be produced. It would result in production of poor quality yield.

A- Denaturation, B- Annealing, C- extension

Denaturation- in this step double stranded DNA is denatured at high temperature of 94⁰C for 15 seconds.

DNA strands separates, and act as template.

Annealing-Primers are added to anneal. This is done at 45⁰ C using Mg²⁺ ions.

Extension- Taq polymerase is used to extend the primer by adding nucleotides at a temperature of 72⁰C.

A- Bam HI

B- Pst I

C- amp^R

Insertional inactivation of antibiotic markers is along and cumbersome process, because it requires simultaneous plating of two culture media. On the other hand chromogenic inactivation is a short method to distinguish between transformants and non transformants.

In chromogenic inactivation, the gene of interest is inserted in between the chromogenic gene.

Therefore, the recombinant DNA would not produce colour due to inactivation of the chromogenic gene. Conclusion:

Colonies producing colour are identified as non transformants whereas the colonies which do not produce colour are transformant colonies.

•Agrobacterium tumefaciens is known to cause infection in plants especially in dicots.

•The Ti plasmid of this bacterium induces tumour formation inside the plant cell.

•The DNA of Ti plasmid gets incorporated with the host cell DNA.

•Recombinant DNA dictates, to form the substances which are necessary for the growth of bacteria.

•However, the tumour causing gene has been deleted by molecular biologist in order to use it as a vector.

Stirred bioreactors are large volume vessels used for large scale production whereas flasks in labs are used to obtain small volume of culture media.