Biochem Plus

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History of Biochemistry:

The roots of biochemistry can be traced back to ancient Greece, where philosophers like Aristotle pondered the composition and processes of life. However, biochemistry as a distinct scientific field began to take shape in the early 19th century. One of the pivotal moments was the discovery of the first enzyme, diastase (now known as amylase), by Anselme Payen in 1833.



Another significant milestone was Eduard Buchner’s demonstration of alcoholic fermentation in cell-free extracts in 1897, which showcased a complex biochemical process outside of living cells. Justus von Liebig’s work in the mid-19th century also laid important groundwork by presenting a chemical theory of metabolism.



The term “biochemistry” itself was first recorded in English in 1848, and by 1903, German chemist Carl Neuberg had coined the term to describe this emerging discipline. The field has since expanded to cover various aspects of cellular components, such as proteins, carbohydrates, lipids, and nucleic acids, and their metabolic pathways.

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Spectroscopy:
– UV-Visible Spectroscopy: Measures the absorption of ultraviolet and visible light by molecules to analyze chromophores like nucleic acids and proteins.

– Infrared (IR) Spectroscopy: Detects molecular vibrations, providing information about functional groups and molecular structure.

– Nuclear Magnetic Resonance (NMR) Spectroscopy: Analyzes the magnetic properties of certain atomic nuclei, giving insights into molecular structure and dynamics.



Mass Spectrometry (MS):
– Matrix-assisted laser desorption/ionization (MALDI) and Electrospray Ionization (ESI) MS: Identify and quantify proteins, peptides, lipids, and other biomolecules based on their mass-to-charge ratio.

– Tandem Mass Spectrometry (MS/MS): Provides sequencing and structural information of peptides and proteins.



Chromatography:
– Gas Chromatography (GC): Separates and analyzes volatile compounds, like fatty acids and small metabolites.

– Liquid Chromatography (LC): Separates and quantifies proteins, nucleic acids, and other biomolecules based on their interaction with a mobile phase and a stationary phase.

– High-Performance Liquid Chromatography (HPLC): A more advanced form of LC that offers higher resolution and sensitivity.

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The Evolution of Industrial Biochemistry

Industrial biochemistry merges principles of biochemistry and industry to innovate sustainable and efficient production methods. The following are key milestones in its evolution:

Foundational Advances:

Enzyme Kinetics and Fermentation: Arthur Harden and Hans von Euler-Chelpin’s Nobel Prize-winning research in 1929 laid foundational knowledge for enzyme kinetics crucial for fermentation processes in industrial biochemistry 

Enzymology: Anselme Payen’s discovery of diastase, the first enzyme, set the stage for enzymology, a core component of industrial biochemistry 

Biotechnological Innovations:

Genetic Engineering: The pioneering work by Paul Berg and Stanley Cohen in genetic engineering and recombinant DNA technology during the 1970s revolutionized biotechnology, enabling the manipulation of genetic materials for industrial applications 3.

Directed Evolution of Enzymes: Frances Arnold’s Nobel Prize in 2018 recognized her contributions to the directed evolution of enzymes, creating tailor-made biocatalysts for specific industrial processes .

Technological Developments:

Enzyme Immobilization: Innovations by Karl Meyer and John Wilder in enzyme immobilization have been pivotal for industrial biocatalysis, enhancing the stability and reusability of enzymes in industrial processes.

Microbial Physiology and Genetics: These advancements have furthered the understanding and application of microorganisms in producing valuable compounds, as evidenced by Alexander Fleming’s discovery of penicillin in 1928.

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Bio-Analytics is an exciting and rapidly evolving field that combines biology, data science, and advanced analytics to unlock the secrets of living organisms and drive innovation in healthcare, agriculture, and beyond. By harnessing the power of big data, machine learning, and cutting-edge computational techniques, researchers in Bio-Analytics are able to analyze vast amounts of biological data and uncover patterns, insights, and breakthroughs that were previously unimaginable.

 

One of the key drivers of Bio-Analytics is the exponential growth in the amount of biological data being generated through technologies such as DNA sequencing, high-throughput screening, and imaging. As the cost of these technologies has decreased and their efficiency has increased, researchers have been able to generate massive datasets that capture the complexity of living systems at an unprecedented scale. However, making sense of this data requires sophisticated analytical tools and techniques that can handle the volume, variety, and velocity of biological data.

 

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Bioanalysis: Fluorescence, Light Microscopy and Microfluidics:

Fluorescence spectroscopy is a powerful analytical technique widely used in bioanalysis to study the structure, interactions, and dynamics of biomolecules. It involves exciting a fluorescent molecule (fluorophore) with light and detecting the emitted fluorescence, which provides information about the local environment and properties of the molecule.



Fluorescence Labeling

To study biomolecules using fluorescence, they are often labeled with fluorescent dyes or proteins. Factors to consider when choosing a fluorescent tag include good absorption, stable excitation, and efficient, high-quantum yield emission. Extrinsic fluorescent dyes can be used to label specific sites on proteins, while intrinsic fluorescence from aromatic amino acids like tryptophan can also be used to probe protein structure and dynamics.



Fluorescence Techniques

Various fluorescence techniques are employed in bioanalysis, including:



– Steady-state fluorescence: Measures the intensity and emission spectrum of a fluorophore under constant illumination.

– Time-resolved fluorescence: Measures the decay of fluorescence intensity over time after pulsed excitation, providing information about the fluorophore’s environment and interactions.

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Best lab Equipments for Biochemistry Students:

1. Micropipettes: Essential for precise liquid handling in small volumes.
2. Centrifuges: Separate mixtures based on density for sample preparation.
3. Spectrophotometers: Measure light absorption to analyze biomolecule concentration.
4. pH Meters: Ensure optimal pH conditions for biochemical reactions.
5. Incubators: Maintain controlled environments for cell cultures and reactions.
6. PCR Machines: Amplify DNA sequences for genetic analysis.
7. Water Baths: Provide consistent temperatures for various biochemical processes.
8. Autoclaves: Sterilize equipment to prevent contamination.
9. Microscopes: Visualize cells and subcellular structures.
10. HPLC (High-Performance Liquid Chromatography): Analyze complex mixtures of biomolecules.

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CRISPR: Mode of Action



CRISPR functions through a combination of RNA and protein components, primarily utilizing the Cas9 protein. The process begins with the introduction of a synthetic guide RNA (gRNA) that is designed to match a specific DNA sequence within the target genome. Once inside the cell, the gRNA binds to the Cas9 protein, which then locates the corresponding DNA sequence and creates a double-strand break. This break can lead to gene disruption or can be repaired in a way that incorporates new genetic material, thus allowing for gene editing.

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What is CRISPR?



CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, is a natural defense mechanism found in bacteria. It allows these organisms to remember and combat viral infections. Researchers have adapted this system into a powerful tool for gene editing, primarily using the CRISPR-Cas9 variant, which employs a guide RNA (gRNA) to direct the Cas9 nuclease to specific locations in the genome. Once there, Cas9 creates double-strand breaks in the DNA, allowing for the removal or addition of genetic material.



Historical Development of CRISPR



The CRISPR system was first identified in the early 2000s, with significant contributions from scientists like Francisco Mojica, who proposed its role in bacterial immunity, and later, Jennifer Doudna and Emmanuelle Charpentier, who adapted it for genome editing. Their work culminated in the development of the CRISPR-Cas9 system, which earned them the Nobel Prize in Chemistry in 2020.

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Common Lab Problems:

Thermal Cycler Malfunctions: Instrument faults may result in no amplification or distorted data, complicating troubleshooting efforts.
Thermal Cycler Malfunctions |solution:

– Regularly maintain and calibrate thermal cyclers according to the manufacturer’s instructions

– Implement a preventive maintenance schedule to identify and address potential issues

– Use a backup thermal cycler or have a service contract with the manufacturer for prompt repairs







Poor Reagent Quality: Using degraded or low-quality reagents can significantly affect experimental outcomes.

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Common Lab Problems:

PCR Failures: Issues with polymerase chain reactions can stem from poor primer design, suboptimal reaction conditions, or contaminated reagents.

PCR Failures| solution:

– Use a high-fidelity DNA polymerase like Q5® High-Fidelity DNA Polymerase from NEB

– Optimize primer design with tools like the NEB Tm Calculator

– Verify the integrity and purity of DNA templates using a NanoDrop spectrophotometer

– Prepare master mixes with ready-to-use reagents like the KiCqStart qPCR ReadyMix from Sigma-Aldrich







Nonspecific Amplification: This occurs when PCR produces multiple bands or smears, often due to mispriming or incorrect annealing temperatures. Nonspecific Amplification| solution:
– Design primers with unique sequences using tools like Primer-BLAST

– Optimize annealing temperatures and times in the PCR protocol

– Use a hot-start polymerase like OneTaq Hot Start DNA Polymerase from NEB

– Titrate primer concentrations to find the optimal level





Inhibition of PCR: Contaminants or inhibitors in the DNA template can lead to reduced amplification efficiency.

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