Introduction
BNG5113
Molecular and Cellular Bioengineering
Spring 2021
Assist. Prof. Elif Eren
Are there any topics you want to study
during this molecular and cellular
bioengineering course?
https://www.menti.com/bcgyqw8sjd The voting code is 4688 5373
bionanotechnology protein covid protein system genomics drug delivery virology engineering in vitro fertilization cancer nanotechnology cellular chemical balance genetic engineering signal transduction genes Q) c gene expression biotechnology gene therapy
Aim of the Course
To provide the molecular and cellular bases of life
from an engineering perspective.
Molecular
Biology
and Genetics
Bioengineering Biomedical Engineering
Week/Place Course Topic To Do Assignments & Deadline W1 March 8, 2021 ONLINE DNA replication • Read the lecture notes of W1 W2 March 15, 2021 ONLINE DNA elasticity • Study lectures of W1 • Read the lecture notes of W2 W3 March 22, 2021 ONLINE DNA sequencing technologies I • Study lectures of W1-2 • Read the lecture notes of W3 W4 March 29, 2021 ONLINE DNA sequencing technologies II • Study lectures of W1-3 • Read the lecture notes of W4 W5 April 5, 2021 ONLINE Melting and other structural transitions in DNA • Study lectures of W1-4 • Read the lecture notes of W5 W6 April 12, 2021 ONLINE DNA nanotechnology • Study lectures of W1-5 • Read the lecture notes of W6 W7 April 19, 2021 ONLINE Driving forces in protein folding and binding I • Study lectures of W1-6 • Read the lecture notes of W7 W8 April 26, 2021 ONLINE Driving forces in protein folding and binding II • Study lectures of W1-7 • Read the lecture notes of W8 W9 May 3, 2021 ONLINE Allostery • Study lectures of W1-8 • Read the lecture notes of W9 W10 May 10, 2021 ONLINE Protein design I • Study lecture of W1-9 • Read the lecture notes W10 W11 May 17, 2021 ONLINE Protein design II • Study lectures of W1-10 • Read the lecture notes W11 W12 May 24, 2021 ONLINE Metabolic engineering • Study lectures of W1-11 • Read the lecture notes W12 W13 May 31, 2021 ONLINE Synthetic biology I • Study lectures of W1-12 • Read the lecture notes W13 W14 June 07, 2021 ONLINE Synthetic biology II • Study lectures of W1-13 • Read the lecture notes W14 The Course
Ø Griffiths, J. B., 1990. Animal Cell Biotechnology, Vol 1-4; Eds. R. E.
Spier, Acad. Pres Inc. Freshney I. R. 1994. Culture of Animal Cells. Wiley Liss Inc. Neumann, K., H., A.Kumar, J. Imani, 2009.
Ø Plant Cell and Tissue Culture – A Tool in Biotechnology; Basics and
Application. Springer – Verlag , Berlin Heidelberg. George, E. F., M.A. Hall, G.-J.De Klerk, 2008.
Reference Books
Evaluation of the Course
Assignment Description Scoring Weight
(%)
Homework A homework will be given. Details will be provided during the course. 100 25
Midterm
The midterm exam will include all subjects covered up to date of the exam. The exact date of the midterm exam will be announced later.
100 35
Final Exam You will have a final exam covering all lectures. 100 40
TOTAL 100 100
DNA Replication
BNG5113
Molecular and Cellular Bioengineering
Spring 2021
Assist. Prof. Elif Eren
1
Outline
Ø PART I: Discovery of DNA
Ø PART II: DNA Structure
Ø PART III: Replication of the DNA
Ø PART IV: Applications in Bioengineering
PART I: Discovery of DNA
12
The discovery of DNA
Mendel
Miescher
Kossel Franklin
Watson/
Crick
1951 1949 1881 1869 1866
“Inheritance”
“Nuclein”
“DNA, A T C G”
“DNA
Visualization”
“Double Helix”
Chargaff
“Equal
amount of A&T, G&C”
13
Gregory Mendel
https://www.sciencelearn.org.nz/resources/2000-mendel-s-principles-of-inheritance
1866
Inheritance in pea plants
Monastery of St Thomas
14
Gregory Mendel
https://www.sciencelearn.org.nz/resources/2000-mendel-s-principles-of-inheritance
1866
Inheritance in pea plants
Traits:
Seed shape, Seed color, Flower color, Pod shape
Round Yellow Purple Inflated Green Wrinkled Green Wh ite Constricted Yellow
15
Gregory Mendel
https://www.sciencelearn.org.nz/resources/2000-mendel-s-principles-of-inheritance
1866
Inheritance in pea plants
Traits:
Seed shape, Seed color, Flower color, Pod shape
Round Wrinkled
16
Gregory Mendel
https://www.sciencelearn.org.nz/resources/2000-mendel-s-principles-of-inheritance
1866
Inheritance in pea plants
Classical genetics:
Study of genetics through the analysis
of the offspring from mating.
17
Friedrich Miescher
1869
Identification of the “Nuclein”
https://www.dna-worldwide.com/resource/160/history-dna-timeline#2
White blood cells
First isolation of
nucleic acid (1869)
Precipitate Nuclein
(DNA associated with proteins)
18
Albrecht Kossel
1881
DNA, nucleotides
https://www.dna-worldwide.com/resource/160/history-dna-timeline#2
Nuclein=
DNA associated with proteins
DNA and RNA are composed of 5 nucleotides:
adenine, cytosine, guanine, thymine, and uracil
19
Erwin Chargaff
1949
A=T, C=G Chargaff’s Rules
1- The amount of A, T, C, G vary among species
2- The number of A is equal to the number of T;
The number of C is equal to the number of G.
20
Rosalind Franklin
1950
Photographs of DNA
https://www.biography.com/scientist/rosalind-franklin
Picture 51
First Picture of DNA
22
Francis Crick James Watson Wurice Wikins
Nobel Prize in Physiology or Medicine 1962
Rosalnd Franklin
The Nobel Prize in Physiology or Medicine 1962 was awarded jointly to Francis Harry Compton Crick, James Dewey Watson and Maurice Hugh Frederick Wilkins “for their discoveries
concerning the molecular structure of nucleic acids and its significance for information
transfer in living material.”
Who discovered:
Who obtained the prize:
23
The discovery of DNA
Mendel
Miescher
Kossel Franklin
Watson/
Crick
Charpentier/
Doudna
2020 1951 1949 1881 1869 1866
“Inheritance”
“Nuclein”
“DNA, A T C G”
“DNA
Visualization”
“Double Helix”
“Genome Editing”
Chargaff
“Equal
amount of A&T, G&C”
24
Emmanuelle Charpentier/ Jennifer Doudna
2020
Genome Editing
CRISPR/Cas9 Gene Editing
sgRNA
DNA
Cas9
sgRNA
DNA
Cas9
sgRNA
DNA
Cas9
25
DNA Sources
Eukaryotic Cells Prokaryotic Cells
Nucleus Mitochondria Chloroplasts
Cytosol
Eukaryote Mitochondria Nuclös
. 'O:uum
26
Bacterial DNA
Staphylococcus pneumonia 2 strains: S type: form capsid, causes pneumonia.
R type: does not form capsid, cleared by
the immune system, does not causes pneumonia.
R
S
Survives
Pneumonia Smooth
Rough
27
Frederick Griffith’s Experiment (1928)
R
S
S
R
S
Survives
Survives
Dies
Dies +
Heat- killed S
Smooth
Rough
28
Bacterial DNA
R
S
Dies +
R
S
Dies +
+ Protease (cleaves proteins)
R
S
+
+ Nuclease (cleaves DNA)
Survives
29
Bacterial DNA
R
S
S
R
DNA
Ø DNA of S type is transferred
to R type bacteria.
Ø The genetic material
transmitted is DNA.
+
PART II: DNA Structure
31
Inheritance in Genetics
Ø Traits are encoded by genes.
Ø Genes are found on DNA.
Ø DNA is organized into chromosomes.
Cell Nucleus Chromosome DNA Gene t of DNA}
Traits
https://www.civilsdaily.com/biotechnology-basics-of-cell-nucleus-chromosomes-dna-genes-etc/
32
Structure of DNA
Ø DNA is a polymer of nucleotides (monomers). Ø Formed by 4 bases:
Purines: Adenine Guanine
Pyrimidines: Cytosine Thymine
Purines Adenine Deoxyribose Guanine Deoxyribose Thymine Deoxyribose Pyrimidines Cytosine Deoxyribose
33
Nucleotide OH —P —O CH2 HOCH2 5 H 3 OH Nucleoside Base A, G, T, orc Position of HO 2 H 3 OH Base 2 H carbon atoms This group is OH in RNA. Phosphate Sugar Sugar This group is OH in RNA.
Structure of DNA
Nucleoside: Sugar + Base Nucleotide: Phosphate + Sugar + Base
Position of
carbon atoms
34
5' end 5' end terminates with phosphate group 5' end Phosphate linked to 5' carbon and to 3' carbon Phosphodiester bonds 3' end 3' end terminates with hydroxyl (—OH) H 5'CH2 o H NH2 HO 3' end H 5'CH2 OH Figure 2.5 Three nucleotides at the 5' end of a single EX'lynucleotide strand. (A) The chemical structure of the sugar—phosphate linkages, showing the 5'-to-3 orientation of the strand (the red numbers are those assigned to the carbon atoms). (B) A common schematic Way to depict a cleotide strand.
Single Strand of DNA
A strand is a polynucleotide.
DNA Strand Formation:
The third carbon of the
deoxyribose sugar forms a phosphodiester bond with the phosphate group of the
following nucleotide.
35
5' end HO 3' end
Single Strand of DNA
Formation of a
Phosphate/Sugar
backbone
Phosphate/Sugar
Backbone
36
5' end 5' end terminates with phosphate group N H2 5' end Phosphate linked to 5' carbon and to 3' carbon Phosphodiester bonds 3' end 3' end terminates with hydroxyl (—OH) 5'CH2 0 5'CH2 5'CH2 HO 3' end Direction: NH2 Figure 2.5 Three nucleotides at the 5' end of a single polynucleotide strand. (A) The chemical structure of the sugar—phcxsphate linkages, showing the 5'-to-3' orientation of the strand (the red numbers are those assigned to the carbon atoms). (B) A common schematic way to depict a polynu- cleotide strand.
Single Strand of DNA
The DNA chain has an
orientation:
5’ end :
Phosphate Group
3’ end:
Hydroxyl Group
Direction:
5’ to 3’
37
Formation of Double Stranded DNA
Ø DNA is formed by
complementary anti- parallel strands.
Ø Base pairing:
(A) Two hydrogen bonds attract A and T. —H —c —H Deoxyribose Adenine Deoxyribose H Guanine T Deoxyribose Thymine Three hydrogen bonds attract G and C. H N c H Deoxyribose Cytosine
38
Formation of Double Stranded DNA
Strand#1:
5’ to 3’
5' end (terminates in S' phosphate) (6 HO S' end (terminates in 3' hydroxyl) S' end (terminates in 3' hydroxyl) S' end (terminates in 5' phosphate) OH p) Figure 2.8 A segment of a DNA molecule, showing the antiparallel orientation of the complementary strands. The overlying blue arrows indicate the 5'-to-3• direction of each strand. The phosphates (P) join the 3' carbon atom of one deoxyribose (horizontal line) to the 5' carbon atom of the adjacent deoxyribose.
Strand#2:
3’ to 5’
39
Formation of Double Stranded DNA
Strand#1:
5’ to 3’
5' end (terminates in S' phos hate) S' end (terminates in 3' hydroxyl) S' end (terminates in 3' hydroxyl) OH o Figure 2.8 A segment of a DNA molecule, showing the antiparallel orientation of the complementary strands. The overlying blue arrows indicate the 5'-to-3• direction of each strand. The phosphates (P) join the 3' carbon atom of one deoxyribose (horizontal line) to the 5' carbon atom of the adjacent deoxyribose.
Strand#2:
3’ to 5’
Phosphate/Sugar
Backbone
Phosphate/Sugar
Backbone
Antiparallel
Strands
40
of O Adenine Thymine Guanine Cytosine Guanine Cytosine Adenine Thymine Phosphate Deoxyribose sugar Base Oxygen Hydrogen Phosphorus C in sugar— phosphate chain Minor groove Major groove 34 Å per complete turn (10 base pairs per turn) D eter C and N in bases Figure 2.6 TWO representations Of DNA, illustrating the three-dimensional structure Of the double helix. (A) In a ribbon diagram. the sugar—phosphate backbones are depicted as bands. with horizon— tal lines used to represent the base pairs. (B) A computer model Of the B form Of a DNA molecule. The stick figures are the sugar—phosphate chains winding around outside the stacked base pairs,
Formation of the Double Helix
The two
complementary
anti-parallel
polynucleotide chain of
DNA are twisted
around one another to form a double helix.
41
TRANSCRIPTION RNA PROCESSING NUCLEUS CYTOPLASM TRANSLATION Nudear envelope DNA mRNA Ribosome Potypeptide
Flow of the Genetic Information
TRANSCRIPTION CYTOPLASM TRANSLATION Ribosome Polypeptide
The Central Dogma
42
Gene 3 DNA template strand mRNA TRANSLATION Protein A u c G c G A U A U A T u c G G c G G Gly G c A T U c c Gene 1 Gene 2 A A Codon Trp Amino acid Ser
The Genetic Code
Second mRNA base 8 0 UUIJ UUC I-JUA I-JUG CUIJ CUC CUA CUG AUC AUG GUIJ GUC GUA GUG Phe Leu Leu lie Met (M) or start Val (V) UCCJ UCC UCA IJCG CCU ccc CCA CCG ACU ACC ACG GCU GCC GCA GCG Ser pro (T) (A) UAU UAC UAA I-JAG CAU CAC CAA CAG AAC AAA AAG GAU GAC GAA GAG Tyr (Y) Stop Stop His (H) Gin Asn (N) Lys (K) Asp (D) Glu UGU Cys UGC UGA Stop UGG Trp(W) CGU CGC Arg (R) CGA CGG AGU Ser AGC AGA Arg AGG GGU GGC Gly (G) GGA GGG z
PART III: Replication of DNA
45
Why do we need to replicate the DNA?
Parental
Cell 2n
Daughter
Cells
2n
Cell Division
Without DNA Replication
Cell Division
After DNA Replication
Parental
Cell
Daughter
Cells
2n n
47
DNA Quantity in the Cell During Cell Cycle
G1
S G2
M
DNA Synthesis
2n, 2C
2n, 4C 2n, 4C
2n, 4C
n= number of complete set of chromosomes
C= DNA content
in the cell
48
DNA Quantity in a Cell
2n, 2C
2n, 4C
1n, 1C 1n, 1C
2n, 2C
2n, 4C
2n, 4C
2n, 2C 1n, 2C
1n, 1C 1n, 1C 1n, 1C 1n, 1C
Egg Sperm
Fertilization
S
G2
Mitosis Meiosis I
Meiosis II
G1
49
DNA Replication
5' 3' 3' 5' (b) First, the two DNA strands are separated. Each parental strand can now serve as a template for a new, complementary strand.
5' 3' 3' 5' 5' 3' 3' 5' (c) Nucleotides complementary to the parental (dark blue) strand are connected to form the sugar-phosphate backbones of the new • daughter" (light blue) strands.
5' 3' 3' 5' (a) The parental molecule has two complemen- tary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.
50
First replication Second replication Parent cell (a) Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix. (b) Semiconservative model. The two strands of the parental molecule separate, and each functions as a template for synthesis of a new, complementary strand. (c) Dispersive model. Each strand of both daughter molecules con- tains a mixture of old and newly synthesized DNA.
DNA Replication Model