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4

GENETICS AND

MOLECULAR BIOLOGY

T

he science of genetics originated when Gregor Johann Mendel published his pioneering work on inheritance in pea plants in 1866 in Proceedings of the Natural History Society of Brünn. But even before that people were aware of inheritance. Farmers used to select seeds of crop plants from those having good traits and even used to adopt breeding techniques to improve the agricultural traits of crop plants. But it was not always a science. Mendel explained his experimental results in the form of laws of heredity. He predicted that there are factors that control each trait, which is transmitted from generation to generation; and that is the subject of the first chapter of this part. It discusses the principles of genetics, the nature of genes and their interaction with environment, genetic recombination, and mutations, their role in variations and evolution, and gene frequencies in a population.

The second chapter mainly deals with the molecular basis of inheritance and the chemical nature and mode of action of genes. It explains the replication of genes, the expression of genes as proteins, and molecular mechanisms of genes regulation. It also discusses the possible involvement of errors in gene regulation and the basis of uncontrolled cell division resulting in the development of cancer.

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FUNDAMENTALS

2.1

OF

BIOCHEMICAL ENGINEERING

%

INTRODUCTION

W

e are all aware of opportunities created by advances in molecular biology. Living cells and their components can be used to produce a large number of useful compounds such as therapeutics and other products. But to obtain significant benefits as a commercial operation, molecular biology needs the support of biochemical engineering. The vital area of biotechnology that is concerned with practical application of biological agents (whole cell systems and biocatalysts) and the methodologies and processes associated with it on an industrial scale is biochemical engineering. Biochemical engineering is applicable in different areas of biotechnology such as biochemical reactions, enzyme technology, environmental biotechnology, microbial manipulations, bioseparation technology, plant and animal cell cultures, and food technology. It consists of the development of new process technology, designing bioreactors, developing efficient, and economically feasible extraction and purification procedures (downstream processing).

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PRINCIPLES

OF

BIOTECHNOLOGY

AND

GENETIC ENGINEERING

n Since the microcarrier culture is well mixed, it is easy to monitor and control different

environmental conditions such as pH, PO2, PCO2, etc. n Cell sampling is easy. n Since the beads settle down easily, cell harvesting and downstream processing of

products is easy. n Microcarrier cultures can be relatively easily scaled-up using conventional equipment

such as fermentors that have been suitably modified.

Because of the many advantages of the technique itself, it has gained great popularity.

Thus, a large variety of microcarriers are available on the market.

FIGURE 20.4 Vero cells cultured on cytodex microcarriers.

6. Fixed-bed reactors. Microcarriers, macrocarriers, or encapsulated beads could be used in fixed-bed reactors. The cells are immobilized in a matrix and the culture fluid is circulated in a closed loop. There is no agitation system. If the bed of immobilized cells is disturbed by the circulating medium, it is said to be a fluidized-bed reactor. Such a process achieves a high degree of aeration and agitation.

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PROTEIN STRUCTURE

AND

ENGINEERING

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The technique of protein fingerprinting involves the following steps:

1. Extract and purify hemoglobin from sickle cell RBC and normal RBC separately in a clean test tube.

2. Digest these proteins with a commercial sample of trypsin separately under standard conditions. Trypsin is another type of serine protease that cleaves the peptide bond adjacent to a lysine or arginine residue in a protein molecule.

3. The cleaved peptides are subjected to paper electrophoresis under pH (pH 2.5) and dry the paper.

4. After electrophoresis they are subjected to paper chromatography perpendicular to the direction of electrophoresis using the solvent system water: butanol: acetic acid in the ratio

5:4:1. The peptides will separate depending on their partition coefficient, which further depends on their degree of hydrophobicity. The more hydrophobic peptide will move fast and the less hydrophobic will move slowly.

5. Remove the chromatographic paper and stain with ninhydrin.

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PRINCIPLES

OF

BIOTECHNOLOGY

AND

GENETIC ENGINEERING

The ability of cAMP to activate expression from the lac operon results from an interaction of cAMP with a protein called CRP (for cAMP receptor protein). The protein is also called CAP (for catabolite activator protein). The cAMP-CRP complex binds to a region of the lac operon just upstream of the region bound by RNA polymerase and that somewhat overlaps the repressor-binding site of the operator region. The binding of the cAMP-CRP complex to the lac operon stimulates RNA polymerase activity 20 to 50-fold. p

p

i

y

z

a

Absence of inducer

repressor binds to the operator region and prevents RNA polymerase from transcribing the operon

repressor mRNA

repressor

p

p

i

o

repressor mRNA

inducer

y

z

a

Presence of inducer

mRNA b-galactosidase

permease transacetylase

(Inactive repressor)

FIGURE 12.12

Regulation of the lac operon in e. coli. The repressor of the operon is synthesized from the i gene. The repressor protein binds to the operator region of the operon and prevents RNA polymerase from transcribing the operon. In the presence of an inducer (such as the natural inducer, allolactose) the repressor is inactivated by interaction with the inducer. This allows RNA polymerase access to the operon and transcription proceeds. The resultant mRNA encodes the β -galactosidase, permease, and transacetylase activities necessary for utilization of β-galactosides (such as lactose) as an energy source. The lac operon is additionally regulated through binding of the cAMP-receptor protein, CRP (also termed the catabolite activator protein, CAP) to sequences near the promoter domain of the operon. The result is a 50-fold enhancement of polymerase activity.

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