Slides for Introductory Biology phase-1.pptx

Sleep Sleep Sloop Sloop

1. Introductory Biology for Engineers SBL100 Prof. Tapan K. Chaudhuri School of Biological Sciences, IIT Delhi

2. Topics for teaching Prof. Tapan K. Chaudhuri ( SBL 100 2022-2023 IInd Sem) Fundamental building blocks of life: Protein, Nucleic acid, Carbohydrate, and lipid   II. Biomolecular structure and function; Anomaly in their function as cause of human diseases   III. Experimental techniques in biophysics   IV. Central Dogma of life, exception to central dogma   V. Concept of gene, gene expression, regulation of gene expression  

3. Text Books Biochemistry by Lubert Stryer 4 th or 5 th Edition / Lewin’s Genes X, XI or XII Principles of Biochemistry by Lubert Stryer 6 th or 7 th Edition /

4. Cellular organization Increasing structural complexity Molecules Atom Macromolecules Inanimate organization (Non-living) organelles Cells Tissue Organs Organ system Whole organism Animate organization (Living)

5. Cells Tiny little compartments where life arises from thousand biochemical reactions Cells Structural organization Functional organization Biological Organization Smallest structural, functional and biological unit of life Atoms 0.1nm 1nm 10nm 100nm 1 μ m 10 μ m 100 μ m 1mm 10mm 100mm 100cm 10m 100m biomolecules Viruses Mycoplasma (smallest cell 0.15-0.3 μ m) Bacteria Cell organelles Human sperm and egg Plant and animal cells Frog egg Chicken egg Ostrich egg (largest cell)~10-13cm Multicellular organisms (Plants, animals)

6. A cell’s complete complement of DNA is called its  genome . In  prokaryotes , the genome is composed of a  single, double-stranded DNA molecule  in the form of a loop or circle. In  eukaryotes , the genome comprises  several double-stranded, linear DNA molecules   bound with proteins to form complexes called chromosomes . Each species of eukaryote has a characteristic number of chromosomes in the nuclei of its cells. Human body cells (somatic cells) have  46 chromosomes . A somatic cell contains two matched sets of chromosomes, a configuration known as  diploid . The letter  n  is used to represent a single set of chromosomes; therefore a diploid organism is designated  2 n . Human cells that contain one set of  23 chromosomes are called gametes , or sex cells; these eggs and sperm are designated  n , or haploid . The matched pairs of chromosomes in a diploid organism are called homologous chromosomes. Homologous chromosomes are the same length and have specific nucleotide segments called genes in exactly the same location, or locus. Genes are segments of DNA that code for a specific protein or RNA molecule . An organism’s traits are determined in large part by the genes inherited from each parent, but also by the environment that they experience. Figure : There are 23 pairs of homologous chromosomes in a female human somatic cell. These chromosomes are viewed within the nucleus (top), removed from a cell in mitosis (right), and arranged according to length (left) in an arrangement called a karyotype. In this image, the chromosomes were exposed to fluorescent stains to distinguish them.

7. Pentose sugar + Nitrogenous base Nucleosides Nucleic Acids Molecular repositories of the genetic information Located in nucleus and acidic in nature: Nucleic acid (Richard Altmann; 1889). Nucleic acids are Biopolymers (nucleotide units are monomers linked with covalent bond; i.e. phosphodiester bond). Two types: DNA & RNA Composition A- Pentose (5-carbon sugar) B- Nitrogenous bases C- Phosphate Nitrogenous Bases Py rimidine (Hetrocyclic ring, parent formula: C4H4N2): C ytosine, U racil, and T hymine Purine (Heterocyclic ring comprise of a pyrimidine ring fused with imidazole ring) A denine and G uanine Nucleosides + Phosphoric Acid Nucleotides (Tri or monophoshphates ) Nt1 + Nt2 + Nt3 + Nt4 + Ntn Nucleic acid (DNA or RNA)

8. DNA: D eoxyribo N ucleic A cid Major nucleic acid acting as central repository of genetic information (except in RNA viruses) Double helical structure is held together by hydrogen bonds formed between complementary bases Adenine forms two hydrogen bonds with Thymine Guanine forms three hydrogen bonds with cytosine

9. RNA: R ibo N ucleic A cid Single stranded ( exception double stranded RNA viruses). Carry out the coding, decoding, regulation and expression of genetic information Ribose sugar present. No thymine contains uracil. Messenger RNA (mRNA): carries the copied genetic information about a protein sequence stored in DNA to the ribosomes, the protein synthesis factories in the cell Transfer RNA (tRNA): Small RNA chain of about 80 nucleotides that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation. Ribosomal RNA (rRNA): T he catalytic component of the ribosomes.

10. Proteins Proteins are linear polymers built of monomer units called amino acids Only L amino acids are constituents of proteins Proteins contain a wide range of functional groups Proteins can interact with one another and with other biological macromolecules to form complex assemblies Some proteins are quite rigid, whereas others display considerable flexibility

11. Proteins

12. Insulin protein function and causes of diabetes

13. Genetic Code DNA RNA Polypeptide chain protein base pairs/nucleotides Amino acids 4 types 20 types Genetic Code / Codons Triplet in nature (composed of three base pairs). Non-overlapping, comma less, non-ambiguous (codes similar amino acid every time) and universal in nature. Polar (read from 5’-P to 3-OH direction). Degenerate in nature (multiple codon same amino acid). A universal start codon ATG (encodes for methionine). Three stop codons :TAG, TAA, TGA (UAG, UAA, UGA) dictates the amino acid identity and order Singlet nucleotide Doublet nucleotide Triplet nucleotides 4 amino acids only 4 x 4 16 amino acids 4 x 4 x 4 64 amino acids

14. What is ‘Central Dogma’ ? The transfer of information from nucleic acid ( eg. DNA, RNA) to nucleic acid, or from nucleic acid to protein may be possible, but transfer from protein to protein, or from protein to nucleic acid is impossible. Information means here the precise determination of sequence, either of bases in the nucleic acid or of amino acid residues in the protein. This was proposed by Francis Crick in 1958 after publishing the double-helix structure of DNA in 1953 with James Watson. By 1952 it was evident that DNA is genetic material with some exceptions.

15. DNA Replication DNA stores genetic information Information is duplicated by replication and is passed on to next generation

16. Transcription DNA is divided into genes Transcription yields a ribonucleic acid (RNA) copy of specific genes Transcription of messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA) happens.

17. Transcription Location : Cytoplasm in prokaryotes, Nucleus in Eukaryotes Process: Conversion of DNA information into RNA molecules Enzyme: RNA polymerase carries out the polymerization/ mRNA synthesis process Steps 1- Initiation: binding of RNA polymerase at promoter site with associated transcription factors and enzymes (Helicase) and opening of double stranded DNA; i.e. transcription bubble formation. 2- Escape from promoter site. 3- Elongation: Insertion of complementary bases and lengthening of RNA chain. 4- Termination: RNA synthesis stops and synthesized RNA molecule releases in the cytoplasm. mRNA synthesizes on noncoding strand also known as antisense or template strand. The other strand is called sense strand or coding strand and has the same base sequence as mRNA except in place of uracil it has thymine base.

18. Translation Translation uses information in messenger RNA (mRNA) to synthesize a polypeptide. Proteins are polymers of amino acids. They are molecules with diverse structures and functions. Polymers are made up of units called monomers The monomers in proteins are the 20 amino acids Functions of protein Enzymes – Biological catalysts Transport of small molecules – Albumin and haptoglobin Transport of oxygen – hemoglobin and myoglobin Membrane proteins – to assist in support Channels in membranes – to allow the passage of molecules or ions Electron carriers in electron transport in the production of ATP

19. Translation mRNA code to synthesis of polypeptide chain Location: cytoplasm of cell Components mRNA: provides the genetic information needed to be translated. Ribosome : macromolecular machine where peptide synthesis happens. tRNA (Transfer RNA): carries the amino acids to ribosome-mRNA complex Amino acid-1 Amino Acid-2

20. Translation tRNA needs to be activated / charged with corresponding amino acids before taking part in translation process. Utilizes specific tRNA activating enzyme specific to the amino acid known as Amino acyl tRNA synthetase . Free amino acyl-tRNA synthetase

21. Protein synthesis on ribosome (Translation)

22. Why protein is not directly synthesized by translating DNA? DNA is the genetic material not RNA or protein. So, by all means DNA is the most valuable part of a cell, if by any chance DNA gets damaged then there will be defected progeny, the biological meaning of life ( ie survive and reproduction) will be lost. Now think, if you will use your main book each time to make 100 photocopies to give 100 students then the main book will be damaged. Instead of this if you use first 10 photocopies to make remaining 90 photocopies then the damage to the main book will be very less or nothing compared to the above method.

23. Exception to Central Dogma

24. Reverse Transcription Reverse transcription is the transfer of information from RNA to DNA (the reverse of normal transcription). This is known to occur in the case of retroviruses, such as HIV and the human T-lymphotropic virus (HTLV). Self-replicating stretches of eukaryotic genomes known as retrotransposons utilize reverse transcriptase to move from one position in the genome to another via an RNA intermediate. They are found abundantly in the genomes of plants and animals. Telomerase is another reverse transcriptase found in many eukaryotes, including humans, which carries its own RNA template; this RNA is used as a template for DNA replication. Applications: Anti-viral drugs (Rev. transcriptase inhibitor), RT-PCR based detection of virues

25. RNA Replication RNA replication is the copying of one RNA to another. The enzymes that copy RNA to new RNA, called RNA-dependent RNA polymerases. SARS-CoV-2 replicates this way without having any DNA stage. Some eukaryotes also contain RdRps , which are involved in RNA interference and differ structurally from viral RdRps

26. Direct Translation from DNA to Protein Direct translation from DNA to protein has been demonstrated in a cell-free system (i.e. in a test tube). Extracts from E. coli that contained ribosomes (but not intact cells) could synthesize proteins from single-stranded DNA templates isolated from other organisms ( e,g ., mouse or toad)

27. Functional execution of the genetic information Requires three distinct processes: 1- Transcription: Copying of the genetic information stored in genomic material into messenger molecule’ i.e. mRNA. 2- Translation: synthesis of encoded message into effector molecules; i.e. polypeptide chains. 3- Protein folding: conversion of linear polypeptide chain into distinct three dimensional functional conformation Information Message Assembly product Function

28. Transcription Location : Cytoplasm in prokaryotes, Nucleus in Eukaryotes Process: Conversion of DNA information into RNA molecules Enzyme: RNA polymerase carries out the polymerization/ mRNA synthesis process Steps 1- Initiation: binding of RNA polymerase at promoter site with associated transcription factors and enzymes (Helicase) and opening of double stranded DNA; i.e. transcription bubble formation. 2- Escape from promoter site. 3- Elongation: Insertion of complementary bases and lengthening of RNA chain. 4- Termination: RNA synthesis stops and synthesized RNA molecule releases in the cytoplasm.

29. Genetic Code DNA RNA Polypeptide chain protein base pairs/nucleotides Amino acids 4 types 20 types Genetic Code / Codons Triplet in nature (composed of three base pairs). Non-overlapping, comma less, non-ambiguous (codes similar amino acid every time) and universal in nature. Polar (read from 5’-P to 3-OH direction). Degenerate in nature (multiple codon same amino acid). A universal start codon ATG (encodes for methionine). Three stop codons :TAG, TAA, TGA (UAG, UAA, UGA) dictates the amino acid identity and order

30. CODON TABLE

31. Translation mRNA code to synthesis of polypeptide chain Location: cytoplasm of cell Components mRNA: provides the genetic information needed to be translated. Ribosome : macromolecular machine where peptide synthesis happens. tRNA (Transfer RNA): carries the amino acids to ribosome-mRNA complex Amino acid-1 Amino Acid-2

32. Protein synthesis on ribosome (Translation)

33. Cloning Strategy Requirements Source of the gene Cloning host Cloning vector DNA modifying enzymes and techniques Plant, animal, or microorganisms Bacterial cells ( E.coli most common ), Yeast cells, Animal models (Rats, guinea pig etc) and plants A circular DNA (plasmid) acts as vehicle to carry the gene in host pMB1 ori : Origin of replication (amplification and maintenance of the plasmid in host cells). Amp: Antibiotic resistance gene (here its ampicillin), used for selection purposes MCS: Multiples cloning sites (restriction enzyme cutting site required for gene insertion) Promoter: essential for transcription of the gene and formation of mRNA promoter Restriction Enzymes: Acts as molecular scissors, cuts out the gene of interest and Cloning Vector at MCS Process: restriction digestion Thermostable polymerase: Replication of gene of interest (amplification) Process: Polymerase Chain reaction (PCR) DNA ligase: acts as molecular glue; joins the cloning vector and gene of interest (requires ATP) Process: Ligation

34. Cloning Strategy Jelly Fish( Aequorea victoria ) glows blue in dark due to a protein called green fluorescent protein (GFP) Isolate genomic DNA GFP gene Restriction enzymes Amplify by PCR reaction Cloning Vector Restriction enzyme treatment Ligation Successful ligation Non-ligated vector Self ligated vector To avoid self ligation digested vector is treated with alkaline phosphatase which removes the 5 terminal phosphate from linearized vector and no self ligation could happens

35. Molecular cloning Molecular cloning Gene Cloning Generation of identical gene copies for expression and regulation purposes Biotechnological, therapeutical, industrial and research applications Also known as Cloning: A process of making identical copies Cloning Gene copies Engineered biological systems Basic Research genes Proteins In Industries (Leather, soap, detergent, food processing etc.) Basic Research on proteins Therapeutics (Insulin, Clot busters, digestive syrups) Bioremediation Transgenic Animals Engineered microorganisms Transgenic Plants Drought resistance, Rich nutrient, Pest resistance etc. Gene therapy Therapeutic protein production, More meat, wool production

36. Cloning Strategy ( Continued… ) Ligation Reaction Transformation in E.coli Non-transformed cells Cells containing recombinant vector Cells containing Non-recombinant vector Selection of transformed cells Antibiotic containing Nutrient Agar plates Antibiotic resistant positive E.coli colonies containing the plasmid DNA

37. A  s chematic representation of a typical blue-white screening procedure A schematic representation of a typical plasmid vector that can be used for blue-white screening For screening the clones containing recombinant DNA, a chromogenic substrate known as X-gal is added to the agar plate. If β- galactosidase is produced, X-gal is hydrolyzed to form 5-bromo-4-chloro-indoxyl, which spontaneously dimerizes to produce an insoluble blue pigment called 5,5’-dibromo-4,4’-dichloro-indigo. The colonies formed by non-recombinant cells, therefore appear blue in color while the recombinant ones appear white. The desired recombinant colonies can be easily picked and cultured. The presence of lactose in the surrounding environment triggers the lacZ operon in  E. coli . The operon activity results in the production of β- galactoisdase enzyme that metabolizes the lactose. Most plasmid vectors carry a short segment of lacZ gene that contains coding information for the first 146 amino acids of β- galactosisdase . The host  E. coli  strains used are competent cells containing lacZΔM15 deletion mutation. When the plasmid vector is taken up by such cells, due to α-complementation process, a functional β- galatosidase enzyme is produced. The plasmid vectors used in cloning are manipulated in such a way that this α-complementation process serves as a marker for recombination. A multiple cloning site (MCS) is present within the lacZ sequence in the plasmid vector. This sequence can be nicked by restriction enzymes to insert the foreign DNA. When a plasmid vector containing foreign DNA is taken up by the host  E. coli , the α-complementation does not occur, therefore, a functional β- galactosidase enzyme is not produced. If the foreign DNA is not inserted into the vector or if it is inserted at a location other than MCS, the lacZ gene in the plasmid vector complements the lacZ deletion mutation in the host  E. coli  producing a functional enzyme.

38. Inoculate liquid broth and grow Recombinant protein production in bacteria Step-A: Recombinant protein expression and harvesting of cells Transformation in E.coli Cells 37 o C shaking condition Add Inducer (IPTG) for protein overexpression Grow further for 4-6 hours Harvest cells High speed Centrifugation (> 10,000 x g for 5-15 min) Cell Pellet (protein expressed intracellularly)

39. Resupend in Lysis Buffer Recombinant protein production in bacteria Step-B: Cell Lysis and Purification Purify the protein using Chromatographic technique Collect Supernatant Cell pellet Lyse cells by sonication Clarify by centrifugation Step-C Screening for identity of the protein Gel Electrophoresis PAGE HPLC or Gel filteration