eChapters

ENZYMES

Enzymes are the heart of Biochemistry

Protein based catalysts.

Enormously effective catalysts: typically enhance rates by 10⁶ to 10¹² fold.

Operate under mild conditions: 0 - 100 °C (or perhaps even 300+ °C for some bacteria in deep ocean), 20 - 40 °C for most organisms; and low pressures (atmospheric).

Very specific: Generally catalyze reaction for a very restricted group of molecules, sometimes for a single naturally occurring molecule of a single chirality.

Introduction
The science of epigenomics has emerged as a key area of research across numerous biological disciplines. Epigenomics is the analysis of epigenetic changes occuring across many genes in a cell or entire organism, whereas the concept of epigenetics focuses on processes that regulate how and when certain genes are turned on and turned off. Understanding on the science of epigenetics can be traced back to the early hypothesis proposed by Aristotle to disprove the theory of preformation. Waddington’s initial conceptualization of epigenetics as a “conceptual tool” to integrate genetics and embryology established a foundational framework for subsequent research. Over time, advancements in molecular biology, developmental biology and evolutionary biology have since profoundly expanded and refined our understanding of epigenetics. Waddington further progressed in the 20th century with his introduction of the “Epigenetic Landscape” model (Baedke, 2013), marking a crucial milestone in the evolution of epigenetic theory.
In modern times, epigenetics refers to the study of heritable changes in gene expression or cellular phenotype that do not involve alterations to the underlying DNA sequence. Recent genetic and biochemical research has improved the understanding of various epigenetic regulation by three mechanisms including DNA methylation, histone modifications and non-coding RNA regulations. These mechanisms induce changes in chromatin conformation (active-open or repressiveclosed), which influences the gene expression that results in gene activation or gene silencing (Fig.1).
The transition between open and closed chromatin states, along with interactions among various epigenetic mechanisms, is essential to maintain optimal cellular function in diverse cell types, tissues and in response to varying developmental or environmental stimuli (Allis and Jenuwein, 2016).
Extensive studies in Arabidopsis led to a profound understanding of epigenetic mechanisms in plant growth and development. Later, intensive investigations was

Introduction

Hydrogels are loosely cross-linked, highly hydrophilic, organic polymers. They can absorb and retain aqueous fluids up to 500 times of their own weight (Singh et al., 2011). In the field application, such absorbents exhibited a limited swelling capacity within the soil matrix.

1. pH meter

pH meter/electrode systems are the most common method of measuring pH in the biology laboratory. Although more expensive and more complicated to use than chemical indicators, pH meters provide greater accuracy, sensitivity, and flexibility. When used properly, pH meters can measure the pH of a solution to the nearest 0.1 pH unit or better, and they can be used with a variety of samples.

1. pH Meter

pH meter/electrode systems are the most common method of measuring pH in the biology laboratory. Although more expensive and more complicated to use than chemical indicators, pH meters provide greater accuracy, sensitivity, and flexibility. When used properly, pH meters can measure the pH of a solution to the nearest 0.1 pH unit or better, and they can be used with a variety of samples.

2. UV Spectrophotometer

Perhaps there is no other technique except UV-visible spectroscopy, which has been so extensively employed and has such a profound impact on progress in biochemical investigations. UV-visible spectroscopy has undoubtedly become an indispensable tool and is routinely used for identification and quantitative estimation of most of the biomolecules. Besides other applications, most of the enzyme assays and the screening of biomolecules recovered in different fractions by separation processes such as column chromatography, density gradient centrifugation etc, rely heavily on spectrophotometer or colorimetric technique.

Escherichia coli is an important model organism for studying microbial metabolism, metabolic regulation, and metabolic engineering. This chapter introduces the use of metabolomics to investigate the metabolic architecture and physiological responses of E. coli. It examines metabolite profiling, central carbon metabolism, metabolic pathways, environmental responses, and metabolic flux analysis. The chapter explains how metabolomic data can reveal changes in cellular metabolism and support the identification of pathway bottlenecks and regulatory mechanisms. Applications in microbial biotechnology, recombinant production, metabolic engineering, and optimization of fermentation processes are considered. The chapter demonstrates how E. coli serves as a useful experimental system for connecting metabolite changes with cellular function.

The functional diversity of chemicals within plants is best demonstrated by terpenoids. More than 30,000 terpenoids have been identified, each one composed of 5-carbon (prenyl diphosphate) building blocks added together to make 10-carbon, 15-carbon, and even 2000–500,000-carbon chains, as is the case for rubber. Terpenes are functionally diverse, and many are integral to primary metabolism, e.g., hormones such as gibberellins and abscisic acid, electron carriers such as plastoquinone and ubiquinone, terpene-derived compounds that form structural parts of membranes such as phytosterols, and photosynthetic pigments such as carotenoids.

Plant terpenes are also available as essential oil or ethereal oil. Essential oil is defined as the volatile oil obtained by the steam distillation of plants. There is a distinction between fixed or fatty oils and volatile oils. Their volatility (boiling point) and plant origin are characteristic properties of these oils.

The establishment of a technology transfer office is an important and complex process for an organization. It needs careful planning to ensure its long-term sustainability. Before establishing a TTO, it is essential to evaluate whether institution’s core mission includes research commercialization, and if the volume and quality of its research output justify the need for an inhouse TTO. For institutions that may not meet all criteria, those institutions can collaborate or outsource for TTO services. It is also important to decide about its operational scope, funding, management, and for what kind of issues the office should develop a policy. There is no “right” way to set up a TTO, but successful TTO does require considering some key issues. This chapter outlines key steps and considerations for establishing a TTO. It also explains what physical infrastructures are needed, manpower, outlines the responsibilities and powers of TTOs.
2.1 Evaluating the need for a TTO
Establishing a TTO is an important step for any research focused organization. But before initiating a planning process for a new TTO, the organization must review its relevance by considering a few fundamental points:
Key considerations for institutions
1. Is institution’s mission focused on “research commercialization” If an institute’s primary mission is education, or it does not support research it might not be justified to create a TTO. For such institutions, it usually makes better sense to seek alternative solutions to the occasional technology transfer without the expense of a formal office. But institutions with strong research commercialization focus, a TTO has the ability to contribute meaningfully to the mission. The Association of University Technology Managers (AUTM), USA has identified following key motivations for initiating academic technology transfer:

When there is indication of linkage from F or testcross data through significant deviation from 9: 3: 3: 1 and 1: 1: 1: 1 ratio, it is implied that independent assortment is not taking place and this is taken as an indication of linkage. The inverse of linkage strength is the recombination value and is often symbolized by ‘p’ whose maximum value is 50% because out of four strands of a tetrad, two are always parental types and two are recombinant types when all the cells show crossing over.

Principle

In this method the estimation of pentose sugar is carried out. Acid hydrolysis of RNA releases ribose (a pentose sugar) which in the presence of strong acid undergoes dehydration to yield furfural. Orcinol, in the presence of ferric chloride as a catalyst, reacts with furfural producing a green colored compound which can be quantitatively estimated at A_{665 nm}. DNA gives a limited positive reaction with Orcinol test.

Materials and Reagents

1. Spectrophotometer
2. Boiling water bath
3. 5% chloroform (HClO₄)
4. Standard RNA solution : Dissolve yeast RNA (500ìg/ml) in 5% HClO₄ . make different dilutions to obtain solutions containing 100 – 500ìg RNA/ml with 5% HClO₄ .
5. Orcinol reagent : Dissolve 100mg of ferric chloride (FeCl₃.6H₂O) in 100 ml of concentration HCl and then add 3.5 ml of 6% solution of Orcinol prepared in alcohol.

Introduction
The use of animals in scientific research has been a cornerstone of medical advancements for decades. However, this practice has also been the subject of intense ethical debate. This lecture explores the necessity of animal experimentation, the ethical considerations involved, and the regulatory frameworks in place to ensure the humane treatment of laboratory animals.
The Need for Laboratory Animals
Animal experimentation has played a crucial role in numerous medical breakthroughs. The importance of laboratory animals in research cannot be overemphasized. Among the various purposes for which humans use animals, experimentation involves relatively few animals and serves a noble purpose in advancing scientific knowledge and human health.
Laboratory animals are invaluable in the drug development process for several reasons:
• Similarity to human organ systems, metabolism, and enzyme functions.
• Suitability as alternatives to human subjects.
• Small size and ability to be reared in controlled environments.
• High reproductive rates.
• Potential for genetic purity, leading to reproducible and reliable results.
The Importance of Animal Welfare
While the benefits of animal experimentation are clear, it’s equally important to consider the ethical implications of using animals for research. The concept of animal welfare is embedded in the Indian Constitution, which states that it is the duty of every citizen “to protect and improve the natural environment including forests, rivers, lakes and wildlife, and to have right compassion for the living creatures” (Article 51 A (g)). This ethical consideration for animals has been emphasized by philosophers, spiritual leaders, and scientists throughout history:

Summary

Human activities involving rapid industrialization and urbanization without concomitant environmental planning have resulted in a situation where substantial parts of our land and water resources have been polluted with organic and inorganic toxicants.  Environmental engineering principles have been utilized to detoxify polluted areas; however, the efforts, adopting predominantly physico-chemical methods, have addressed the issue to a restricted extent, focusing on solutions for specific categories of pollutants and applicable for limited areas, in addition to being expensive. Hence cost-effective sustainable alternatives in the form of bioremediation (involving microorganisms) and phytoremediation (involving plants alone or along with associated rhizospheric microorganisms) are gaining public acceptance. Considerable scientific evidence has proved that many plant species can detoxify the pollutants utilizing various mechanisms. In this background, in vitro plant cultures have served as invaluable tools in identifying appropriate plant species for phytoremediation. Better understanding of the complex interactions underlying phytoremediation has been facilitated using in vitro cultures where the only variable is the pollutant, in contrast to field experiments where there are many confounding variables. While straightforward extrapolation of in vitro data can be erroneous, these systems provide the basic framework for translating laboratory-scale experiments to field-scale technologies.

Performance metrics and indicators
Traditionally, in the past, TTOs’ performance was measured in monetary terms, such as universities’ revenues derived through commercialization. The methodology, however, is prone to shortcomings, in light of the fact that various TTOs do not receive significant amounts of financial proceeds through their technology transfers. In addition, relying on income does not depict a TTO’s true potential, in light of the fact that it does not account for the total range of outputs of research and commercialization opportunities. Recognizing these limitations, academics have found innovative ways to measure TTO efficiency, such as monetary and non-monetary indicators. Financial indicators typically entail measurable outcomes such as number of signed licenses, income from licenses, royalties collected, equity income, and number of spin-offs or startups created. These are actual gauges of the economic returns from commercialization efforts, and in the majority of instances are used in the context of benchmarking the performance of a TTO in a comparative context, perhaps against peer institutions or industry averages. Non-financial indicators consist of royalty distributions to academics, having an integral role in encouraging academics to participate in commercialisation activity. By incorporating monetary and non-financial metrics, universities might have an enhanced, comprehensive evaluation system for their TTOs. Such evaluation is sure to ensure assessment extends beyond short-run monetary metrics and looks to incorporate several organization, institutional, and research-oriented determinants in driving successful technology transfer.

Origin of Species

A biological species is a group of reproductively isolated individuals

Speciation usually occurs via a branching process called cladogenesis

Divergent evolution can be allopatric, parapatric, or sympatric

Müntzing was able to re-create an allotetraploid species

Evolution can proceed gradually or be punctuated by periods of rapid change

In this chapter, the focus is on understanding the importance of experimental design and being familiar with key terminology. You’ll also learn about the three fundamental principles of experimental design and their roles in conducting experiments. Additionally, you’ll learn how to do randomization of treatments in various experimental designs using R.

Experimental Design

Experimental design is the methodical planning of scientific experiments for reliable results by controlling variables and minimizing biases. It serves as the foundation for effective research. Some key definitions to enhance understanding of this concept:

Key terminologies

• Experiment: Structured investigation for knowledge acquisition or validation.

• Experimental unit: Entity receiving treatment (e.g., land plot, pot).

• Treatment: Specific procedure for assessment and comparison.

• Experimental error: Variation among identically treated units.

Principles of Experimental Design

Experiments are vital tools in agricultural research, aiming to assess treatment effectiveness and quantify their impact. However, extraneous factors can confound results. To mitigate these issues, we depend on three core principles of experimental design: replication, randomization, and local control.

Abstract

Endophytes are microorganisms  that live inside the living plant tissues for at least part of their life without causing any apparent disease symptoms in the host. In general  they  can be divided into two main types namely, obligate and facultative endophytes. Facultative endophytes are capable to survive in the soil, on the plant surface, inside the plants as well as on artificial nutrients and can be cultured away from their host. Endophytes which inhabit inside plant tissues throughout their lifespan are called as obligate endophytes. Natural products produced by endophytic microbes are found to be antimicrobial, antiviral, anticancer, antioxidants, antidiabetic and immunosuppressant. Therefore, endophytes form an untapped source of novel natural compounds for application in agriculture, medicine and industry.

Summary

Expressed sequence tags are small pieces of DNA sequence (usually 200 to 500 nucleotides long) that are generated by sequencing either one or both ends of an expressed gene. Currently various EST sequencing projects are underway for numerous organisms, generating millions of short, single-pass nucleotide sequence reads, accumulating in EST databases. Extensive computational strategies and various databases have been developed to organize and analyse both small- and large-scale EST data for gene discovery, functional annotation of putative gene products as well as transcript and single nucleotide polymorphism analysis. Present chapter provides an overview of the significance of ESTs, their properties, databases, analysis software and the applications of ESTs.

Summary

Genes are discrete elements that govern the appearance of specific traits. Morgan Sturtevant and their coworkers demonstrated that genes have specific address. They reside at particular locations on particular chromosomes and these addresses remain constant from one individual of the species to the next. Transcription is the first step leading to gene expression. It is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes. During transcription, a DNA sequence is read by RNA polymerase, which produces a complementary, antiparallel RNA strand. translation is the second stage of protein biosynthesis (part of the overall process of gene expression). In translation, messenger RNA (mRNA) produced by transcription is decoded by the ribosome to produce a specific amino acid chain, or polypeptide, that will later fold into an active protein.

When Gene Expression Appears to Alter Mendelian Ratios Lethal Allele Combinations

Us recessive lethal alleles eliminaters a progeny class. Multiple Alleles

A gene can have more than two alleles, but a diploid individual only has one or two of them.

Different allele combinations can produce different phenotypes and different severities of symptoms.

Different Dominance Relationships

Incomplete dominance of an allele produces a phenotype in the heterozygote that is intermediate between that of either homozygote.

Codominant alleles are both expressed in a heterozygote. Epistasis-When One Gene Affects Expression of Another

In epistasis, one gene masks the effect of another. An example of epistasis in humans is the Bombay phenotype.

The Bombay phenotype results in O type blood for individuals homozygous recessive for the recessive “h” allele.

There are many different methods of extracting DNA bands from an agarose gel. Which one you choose will probably depend on the consumables you have available in your lab. Another important consideration is the yield/ purity of the DNA after extraction. Agarose contains various impurities which may inhibit downstream reactions if not efficiently removed from the DNA. We used to give the generic term ‘hoonose’ to the unspecified carbohydrates that co-purified along with the DNA and inhibited enzyme reactions. Modern spin-column kits are very good at removing impurities, old-fashioned ‘kitchen sink’ methods are less good.

Cutting Out the DNA Band

Most of the methods require you to cut out the band. You must visualise the band, in an ethidium bromide stained gel, in a dark-room on a UV light- box (a trans-illuminator). UV is dangerous, wear gloves, long-sleeves and face protection. Never look at UV with unprotected eyes. If possible, set the trans-illuminator to long-wavelegth UV (or low-power) and minimise the amount of time the DNA is exposed. This is because the UV mutagenises the DNA at a measurable rate. Use a scalpel blade to cut around the band of interest. Switch off the transilluminator, switch on the white light and carefully remove the band from the gel and place it on the glass. It is good to trim off as much empty agarose as possible so go back to UV illumination briefly to do this. Place the excised band in a 1.5mL microfuge tube.