eChapters

A spectrophotometer is an analytical instrument that measures the absorbance of a test sample (solution). The term “spectroscopy” comes from the word “spectrum”. “Spectroscopy” therefore implies the use of multiple wavelengths of light. Spectrophotometers have the ability to specifically measure absorbance at specific wavelengths. The most commonly used method to allow this involves a “monochromator”, a device (either a prism, or more commonly, a diffraction grating) that splits the incident light into its component wavelengths, and allows only light of the desired wavelength to reach the sample. The ability to measure absorbance at different wavelengths is very useful, because the extinction coefficient of a compound varies with wavelength. In addition, the absorbance spectrum of a compound can vary depending on the chemical composition of the compound, and depending on the nature of the solvent around the compound.

Absorbance is a useful quantity and follows the Beer-Lambert law. The extinction coefficient of a molecule at a given wavelength can be calculated using the Beer-Lambert equation from absorbance measurements for solutions of known concentration, which states that:

Abstract

Biological databases that are composed of a large collection of digital nucleic acid sequences, protein sequences, or other polymer sequences stored on a computer, which is made available to the research community worldwide using web servers. The vast amount of information generated in molecular biology is analyzed, curated, tabulated, and arranged in databases. There are different types of biological databases, based on their contents. Besides, the database management system, a program to manage the databases, can be of two types: distributed DBMS and relational DBMS. This chapter briefly narrates the importance and purpose of biological databases.

Abstract

Molecular phylogeny is an important method for understanding the evolutionary relationships between different organisms. Molecular phylogeny is based on the idea that organisms that share a more recent common ancestor will have more similarities in their DNA or protein sequences. The chapter seems to cover the basic concepts and terminology used in molecular phylogeny, including the difference between rooted and unrooted trees, as well as various methods used for constructing phylogenetic trees. Further, chapter also covers some common tools and software used for analyzing molecular data and constructing phylogenetic trees. Overall, this chapter provides a useful introduction to molecular phylogeny and the methods used for understanding the evolutionary relationships between different organisms. Understanding these concepts is important for many areas of biological research, including evolutionary biology, ecology, and biotechnology.

Autoclave Operating Procedures

Place all material to be autoclaved in an autoclavable tray. All items should have indicator tape. Separate liquids and solids should be autoclaved separately. Ensure that the lids on all bottles are loose. Do not put a large number of items in tray, to ensure that all items reach the appropriate temperature. Also allow sufficient air/steam circulation.

Steps in autoclave

Pressure should read ‘O’ when you open the pressure chamber of the autoclave.

Place items to be autoclaved in the autoclave and close the door. Some autoclaves require that you also lock the door after it’s closed.

Set time - typically 20 minutes.

Temperature should be set at 121°C already, but double-check and change if necessary.

Set cycle: If liquid, set “liquid cycle” or “slow exhaust”. If dry, set “dry cycle” or “fast exhaust” + dry time.

Start the cycle. On some autoclaves, the cycle starts automatically at step 5. On others, turn to “sterilize”

Abstract

PCR primer design is a crucial skill for molecular biologists, as it enables the study of gene expression, gene amplification, and molecular markers. Primer3 is an online tool widely used for designing optimal PCR primers, which are pairs of short oligonucleotides that initiate DNA synthesis in vitro using a thermocycler. This tool uses a range of parameters to generate high- quality primers, including target sequence length, melting temperature, GC content, specificity, and product size. Understanding and effectively utilizing these parameters is essential for achieving accurate and reliable PCR results. In this chapter, the practical aspect of primer designing using the online tool Primer3 has been discussed. The parameters and the purpose of their usage have been given in a tabulated manner.

Storage

Nucleic acids are very sensitive to contaminations hence care should be taken while processing and storing DNA and RNA. The following properties of reagents and conditions need to be taken care of:

Heavy metals cause breakage of phosphodiester linkage. One can use EDTA which is an excellent heavy metal chelator.

UV rays cause extensive damage to nucleic acids. UV light at 260 nm causes breakage of thymine dimers and cross-link, including loss of biological activities.

Ethidium bromide results in photo oxidation of DNA. Oxidation products can cause phosphodiester breakage.

Nucleases are found on human skin; therefore, it is advisable to avoid direct or indirect contact between nucleic acids and fingers.

Most DNAes are not very stable; however, many RNAses are very stable and can absorb to glass or plastic and remain active.

At -20°C: extensive single and double strand breaks results. Hence for long-term storage it should be stores at -70°C. ? It is best to store DNA in high salt ( >1M) in the presence of high EDTA (>10mM) at pH 8.5, for long-term storage.

Storage of DNA in CsCl with ethidium bromide in the dark is an excellent storage medium. There is about one phosphodiester break per 200 kb of DNA per year.

Storage of ë DNA in the phage is better than storing the pure DNA.

Cell Genotypes

A large variety of bacterial strains, like E. coli, are frequently used in molecular biological experiments. Students should choose the strain or strains required for an experiment on the basis of genetic properties of the strains. It is thus pertinent that a sound knowledge of the genetic makeup of the E. coli stains is acquired.

The genetic makeup of the strains of E.coli is distinctly different from the wild type (K12 or B strains) strains from which they are derived and contains genes that can be modified. Genes are supposed to be mutated when they are not listed in a genotype. In most cases, the genes listed are mutated to the point of inactivity. In a few cases, a “-” (indicating inactive or absent or deleted), “+” (indicating active). , The genotype is a factor in choosing a bacterial strain. Chosen strains should be ascertained for their utility for the specific experiment.

Small Scale Cultures

Fresh culture from a freshly streaked plate should be taken while experimenting with E. coli cells. For preparing a small scale culture of E. coli , prepare 3-5 ml of LB medium/broth (or appropriate broth include antibiotic if the culture contains a plasmid) in two sterile 50 ml tubes. Inoculate one tube with a single colony from a fresh plate. The second tube is used as a broth control. Incubate both tubes at 37°C, shaking vigorously overnight. Inspect the tubes the next morning. The broth control should be clear and the inoculated culture should be very turbid. Make a note of any debris found in the tubes and only incubate longer if the culture is not dense. Never allow cells to overgrow. For some applications, cells can be stored at 4°C for short periods prior to use.

Abstract

The Basic Local Alignment Search Tool, or BLAST, is an online tool provided by the National Center for Biotechnology Information (NCBI) that allows users to search for matches between their query sequences and the sequences stored in NCBI databases. BLAST can be used to search for nucleotide sequences against nucleotide sequences, protein sequences against protein sequences, and protein sequences against translated nucleotide sequences in the databases. BLAST is widely used to study sequence similarity, identify target sequences, analyze the functional aspects of molecular sequences, and investigate evolutionary relationships. This chapter details on the BLAST and its applications.

Participatory curriculum development (PCD) in extension education is an emerging approach to help and improve the quality of education in farm universities. The main steps of the PCD approach adopted here have high practical utility for the design of formal course contents for the Undergraduate and Postgraduate courses in Agricultural Extension. A great deal of thinking is required while deciding on how much participation will be required from which stakeholders, at what level(s).

Principle

Apart from PCR for generating large amounts of DNA, the second method is to use bacteria to replicate the DNA and then purify the replicated DNA from the bacteria. Plasmid DNA is usually the DNA of interest for this method. A plasmid is a double stranded circular DNA molecule that will replicate in an organism. A typical plasmid contains four important features:

The plasmid must be circular, because bacteria generally will not replicate linear ? DNA.

The plasmid must contain a sequence that functions as an origin of replication (ori).

The plasmid must contain a selection mechanism that will force the bacteria to retain the DNA; the most common type of selection mechanism used in bacteria is a gene for resistance to an antibiotic such as ampicillin or tetracycline.

The plasmid must contain a region for the insertion of the experimental DNA. A generic plasmid exhibiting these features is shown at right.

An expression plasmid that allows expression of foreign DNA should have a strong promoter segment for transcription of inserted DNA.

Must have an effective ribosome binding site that will allow efficient translation of the transcribed RNA.

Must have high copy number, i.e., the host bacterium should have many copies (usually >100)

Isolation of plasmid DNA from E. coli is a common routine in research laboratories. One can perform a widely-practiced procedure that involves alkaline lysis of cells. This protocol often referred to as a plasmid “mini-prep,” yields fairly clean DNA quickly and easily.

Procedure

Fill a microcentrifuge tube with saturated bacterial culture grown in LB broth + antibiotic. Spin tube in microcentrifuge for 1 minute, and make sure tubes are balanced in microcentrifuge. Dump supernatant and drain tube briefly on paper towel.

Repeat step 1 in the same tube, filling the tube again with more bacterial culture. The purpose of this step is to increase the starting volume of cells so that more plasmid DNA can be isolated per prep. Spin tube in microcentrifuge for 1 minute. Pour off supernatant and drain tube on paper towel.

Add 0.2 ml ice-cold Solution 1 to cell pellet and resuspend cells as much as possible using disposable transfer pipet.

Abstract

Proteins are complex molecules composed of one or more polypeptide chains made up of amino acid residues. These molecules perform a variety of essential functions in living organisms, including DNA replication, molecule transport, catalyzing metabolic processes, and providing structural support to cells. In this chapter, we explore the four levels of protein structure: primary, secondary, tertiary, and quaternary. Primary structure refers to the specific sequence of amino acids in a polypeptide chain, while secondary structure describes the folding of the chain into alpha-helices, beta-sheets, or other patterns. Tertiary structure is the three-dimensional shape of a single polypeptide chain, and quaternary structure refers to the interactions between multiple polypeptide chains to form a larger protein complex. Additionally, this chapter covers various methods used to predict protein structure, including homology modeling, ab initio prediction, and threading algorithms.

Abstract

When only sequence data is available, homology modeling has become a useful tool for predicting protein structure. Protein structure is often more important than sequence data when evaluating protein function. Homology modeling, also known as comparative modeling, is based on the principle that two sequences that exhibit a high degree of identity or similarity will also share similar structures. This chapter explores the steps involved in homology modeling, including template selection, alignment, model building, refinement, and validation. The Ramachandran plot is a valuable tool for assessing the conformation and bond angles in a protein model, and we discuss its significance in the context of homology modeling. Ultimately, homology modeling is an effective method for predicting protein structure and has numerous applications in protein engineering, drug discovery, and other fields.

Technology Scouting and Innovations in Technology Incubation
Technology scouting and innovations in technology incubation have a symbiotic relationship, fuelling each other for the benefit of budding startups and the broader technology ecosystem. Let’s dive deeper into their individual roles and how they interact:
Technology Scouting
Technology Scouting is the process of discovering, analyzing and evaluating new or existing technologies that will help them with their innovation process. In simple terms, it is a process for companies to find the necessary technology outside the company.
Hunting for promising technologies: Scouts actively search for emerging technologies, patents, research advancements, and market trends across various sectors.
Evaluating potential and fit: They assess the technical feasibility, market potential, and compatibility with the needs of incubators and their supported startups.
Connecting innovators and ventures: Scouts bridge the gap between technology creators (researchers, inventors, startups) and potential users (incubators, established companies).
Fuelling innovation pipelines: Technology scouting helps incubators access a wider range of technologies and ideas, enriching their portfolio and diversifying the innovations supported.

Solution Preparation

1. Glycerol mix (1 L)

Weigh 25 grams (liquid weight) of glycerol and

Add dH₂O to 1 Liter (Autoclave).

2. Potassium mix (1 L)

170 mL of 1M KH₂PO₄ (monobasic),

720 mL of 1M K₂HPO₄ (dibasic), and

110 mL of dH₂O (Autoclave)

Viruses are much simpler than cells

Protein capsid

Genetic information stored on DNA or RNA:

Types of genome:

Double strand DNA: sometimes circular

Single strand DNA: sometimes circular

Double strand RNA

Single strand RNA

Some can be crystallized

Preparation of Solutions

GTE buffer

50 mM Glucose,

25 mM Tris-Cl,

10 mM EDTA,

pH 8

NaOH/SDS lysis solution

0.2 M NaOH,

1% SDS

Abstract

Systems biology is an interdisciplinary field that combines computer and mathematical analysis with biological research to understand the complex interactions within biological systems. This chapter explores the history and evolution of systems biology and its significant impact on modern biology. Systems biology uses a holistic approach to analyze biological systems at the molecular, cellular, and organismal levels, providing a better understanding of biological processes and the interactions between genes, proteins, and other molecules. Systems biology has immense potential in improving human health, agriculture, and the environment, and this chapter explores the ongoing efforts to translate systems biology research into practical applications.

Reagents

EDTA

Ethanol, absolute

Isopropanol

Phosphate Buffered Saline (PBS), 1X

Proteinase K

Sodium Acetate, 3 M pH 5.2 (Molecular Biology Grade)

Sodium chloride (NaCl)

Sodium Dodecyl Sulfate (SDS ), 10%

Tris EDTA (TE), pH 7.4

Tris EDTA (TE), pH 8.0

Tris HCl, pH 8.0

Q.1.    What is progeny row trial ?

    Answer
    
    The replicated experiment designed to compare a number of parent plants with respect to any characteristic under study is known as progeny row trial.