1
Q
Atoms
A
- both living and nonliving things are made up of atoms
- water, bacteria, humans
- atoms react with each other and make molecules -> organelles -> cell -> tissue -> organ -> organ systems -> organism
2
Q
structure of atoms
A
- proton +
- neutron
- electron -
- proton + neutron = nucleus
- nucleus is positive
- electrons orbit nucleus
- # protons = # electrons
- atoms are neutral (charges cancel)
3
Q
electrons
A
- orbit around nucleus
- arranged in shells
- electron shell- space were electrons are located around the nucleus
- shell 1- max of 2e-
- shell 2- max of 8 e-
- shell 3- max of 8 e-
- most atoms do not have a max # of electron in their outermost shell -> unstable
- unstable atoms will interact to form stable shells
- form chemical bonds to form molecules
4
Q
ionic bond
A
- atoms gain or lose electron
- NaCl
- ion atom- gained or lost e-
- sodium has a single e on outermost shell -> loses electron
- chloride is missing one e on outermost shell -> gains electron
- they complete each others shells
- Na becomes +
- Cl becomes -
- attraction is the ionic bond
5
Q
covalent bond
A
- atoms get together and share electrons
- electrons orbit around the nucleus of both atoms
- common
6
Q
hydrogen bonds
A
- water molecules are held together with hydrogen bonds
- H2O has covalent bonds holding the molecule together between atoms
- electrons are not shared equally
- electrons stay closer to O bc its larger and has more protons
- O is slightly neg
- H is slightly positive
- polar molecule
- heat breaks down H bonds -> water evaporates
7
Q
water: solvent
A
- a good solvent
- NaCl goes into solution readily (dissociates, ionizes)
- play an important role in chemical rxn that take place in cell
- dehydration synthesis
- hydrolysis
- water surrounds NaCl and pulls the charges apart -> forms ions
8
Q
acids, bases, and salts
A
- acids ionize- H+ and a negative ion
- HCL -> H+ + Cl-
- bases ionize -OH- (hydroxide ion) and a positive ion
- NaOH -> Na+ + OH-
- salt- positive ion and a negative ion (not H+ or OH-)
- NaCl -> Na+ + Cl-
9
Q
pH
A
- hydrogen ion concentration
- 0-14
- 7- neutral -> H+=OH-
- acid- <7
- base >7
- household bleach- basic
10
Q
carbohydrates
A
- C, H, O
- 2 to 1 ratio of hydrogen to oxygen
- monosaccharides- simple sugars
- glucose C6H12O6
- glucose is main source of energy in cells
- ribose- one of the molecules found in RNA
11
Q
disaccharide: dehydration synthesis
A
-sucrose- made up of fructose and glucose -> sugarcane
-glucose and fructose combine through dehydration synthesis
-produces a H2O as a result
-
12
Q
hydrolysis
A
- breaking down molecules
- sucrose -> fructose and glucose
- water is used in the rxn
13
Q
lactose
A
- disaccharide
- milk sugar
- made up of glucose and galactose
14
Q
maltose
A
- disaccharide
- made up of two glucoses
- breakdown product of starch
15
Q
polysacchrides
A
-made up of many units of simple sugars polymers of glucose: -cellulose- plant cell wall -glycogen- glucose is stored in animals -starch- glucose is stored in plants
16
Q
lipids
A
- C, H, O
- there is no 2 to 1 ratio
- simple lipids- triglyceride
- triglyceride- made up of glycerol and 3 fatty acids
- ^ energy storage molecules
17
Q
triglyceride
A
- glycerol is the vertical portion
- 3 fatty acids attach to the glycerol through ester linkages
18
Q
phospholipids
A
- plasma membranes
- organelles
- made up of glycerol and two fatty acids and phosphate
- glycerol and phosphate make up the hydrophilic head
- fatty acids -> hydrophobic tail
- form a bilayer -> fatty acids in the interior
19
Q
proteins
A
- C, H, O, N, S
- building blocks- amino acids
- 20 different amino acids
20
Q
amino acid
A
- amino group- NH2
- carboxyl group- COOH
- side group- R group
- alpha carbon
21
Q
peptide bond
A
- amino acids form polypeptides through peptide bonds
- C of carboxyl group and N of amino group
- C-N bond
- water product
22
Q
protein structure
A
- primary- amino acid sequence of polypeptide chain
- secondary- twisting & folding of the polypeptide chain -> due to hydrogen bonds
- tertiary- disulfide bonds is formed between diff parts of the polypeptide -> 3D shape
- quaternary- two or more polypeptide chains interact to make a functional protein/enzyme
23
Q
hemoglobin
A
- polypeptide chains
- polypeptides have a specific amino acid sequence
- valine-histidine-leucine-glutamic acid
- sickle cell anemia -> diff sequence -> valine takes on glutamic acid spot
- shape of the protein changes
- RBCs sickle shaped
- not flexible- trouble getting through the capillaries
- health problems
24
Q
Adenosine triphosphate
A
- ATP
- three phosphates
- adenine and ribose = adenosine
- when terminal phosphate is removed -> energy is released and used
- quick source of energy in cells
- energy carrier molecules
- synthesis, movement, transport
- ATP -> ADP + phosphate + energy
- ADP + phosphate + energy (comes from catabolic processes) -> ATP
25
metabolism
-all the chemical rxn that take place in a cell
26
catabolism
- metabolic process
- larger molecules are broken down into smaller molecules
- break down process
- cellular respiration- glucose is broken down in to CO2 and H2O
- release energy
- energy released is stored in ATP -> used to make ATP
27
anabolism
- metabolic process
- synthetic process
- larger molecules are synthesized from smaller molecules
- photosynthesis
- CO2 and H2O are used to make glucose
- energy is used
28
enzymes
- constantly taking place
- depends on enzymes
- biological catalysts
- speed up chemical rxn
- come out of rxn unchanged
- not used up
- in absence of enzymes- cells cannot survive bc rxn are so slow
- specific for substrate
- substrate- substance with which the enzyme react
- speed up chemical rxns by bringing molecules together so they can react with each other so a larger molecule is synthesized
- some weaken chemical bonds in molecules -> molecule is broken down
29
denaturation
- temperature- at high and low temperature enzymes are slow
- pH
- substrate concentration- as substrate increases enzyme activity increases until it can not longer increase -> levels off
- proteins lose 3D shape -> no longer functions
30
enzyme inhibitors: competitive
- competitive
- competitve- compete with the substrate for the active site on the enzyme
- ex. sulfanilamide- synthetic drug- UTI
- converts para aminobenzoic acid (PABA) to -> folic acid
- drug takes the place of PABA on the enzyme
- inactivates the enzyme
- bacteria needs folic acid to reproduce so the inhibitor will kill the bacteria
31
enzyme inhibitors: noncompetitive
- binds to the allosteric site on enzyme
- allosteric site- site other than the active site
- shape of the active site is changed
- enzyme is inactivated
- cyanide
32
cellular respiration
- glucose is catabolized
- oxidation reduction rxn
- loss of electron or hydrogen atom- oxidation
- gain of electron of hydrogen atom- reduction
- leo says ger
- these rxns are coupled
- organic molecules are oxidized
- NAD+- coenzyme/electron carrier picks up the H+ (reduced) -> NADH
33
catabolism of glucose
- energy is released
- energy is used to make ATP from ADP and phosphate
- cellular respiration- glucose metabolism
34
aerobic respiration
- O2 is used
- most common
- C6H12O6 + 6O2 > 6CO2 +6H2O + energy
- glucose is oxidized to CO2 -> glucose loses all 12 H atoms
- O2 reduced to water -> picks up the H glucose lost
- glucose is not directly converted to CO2 and water (too much energy would be released)
- extracts energy from glucose a little at a time
- involves glycolysis, transition rxn
- krebs cycle, oxidative phosphorylation (electron transport chain)
35
glycolysis
- sugar splitting
- takes place in cytosol (liquid part of cytoplasm)
- conversion of glucose to glucose phosphate -> uses an ATP
- carries out 7 different rxn before it gets ATP out of glucose
- products: 2 pyruvic acid + 2 NADH + 4ATP
- net gain of 2 ATP (2 ATP were used during glycolysis)
- substrate level phosphorylation- phosphate is added from a substrate to ADP
36
glycolysis
- sugar splitting
- takes place in cytosol (liquid part of cytoplasm)
- carries out 7 different rxn before it gets ATP out of glucose
- products: 2 pyruvic acid + 2 NADH + 4ATP
- net gain of 2 ATP (2 ATP were used during glycolysis)
- substrate level phosphorylation- phosphate is added from a substrate to ADP -> makes ATP
- phosphate and energy are directly transferred from a substrate ADP to make ATP
- 10 different rxns 10 different enzymes in glycolysis
37
transition reaction
- pyruvic acid goes into transition reaction
- takes place in matrix of mitochondria
- pyruvic acid is oxidized and decarboxylated -> acetyl CoA
- NAD+ is reduced to NADH
- CO2 is released from pyruvic acid as a waste product of aerobic respiration (exhale)
- each molecules of glucose -> 2 acetyl CoA + 2NADH + 2 CO2
- Acetyl CoA goes into krebs cycle
38
Krebs cycle
- takes place in matrix of mitochondria
- reactant- 2 acetyl CoA + 2NADH + 2 CO2
- product- 6NADH + FADH2 +4CO2 + 2ATP
- Acetyl CoA comes out of transition rxn and reacts with oxaloacetic acid -> makes citric acid
- NAD+ is reduced to NADH
- 2 ATP is made by substrate level phosphorylation
- 4 oxidation reduction rxns take place
- CO2 is released as a waste product (exhale)
- NADH and FADH has some of the energy from glucose -> has to extract energy from here to make more ATP
39
electron transport chain
- NADH and FADH interact with the electron transport chain to use the energy they carry from glucose and make it into ATP
- takes place in inner membrane of mitochondria
- in the membrane: flavin mononucleotide (FMN), uniquinone (Q), cytochromes (cyt)
- NADH interacts with the 1st molecules of ETC -> FMN -> NADH is oxidized into NAD+ (first time NADH is being oxidized)
- FMN grabs the H+ and gets released into intermembrane space -> the e- it holds onto has energy
- when e- is moved from one molecule to another energy released and is used to pick up H+ from the matrix and release the H into the intermembrane space
- eventually the e- get picked up by O2 (reduced) -> O2 then reacts with H and makes water -> reduced water -> final electron acceptor
- chemiosmosis- build up of H in the intermembrane space -> diffuse into the matrix through a tiny transport channel hole made by ATP synthase -> rush in with force
- energy from the H+ helps the cell make ATP from ADP and phosphate -> oxidative phosphorylation
- makes 3 ATP molecules per NADH -> there are 10 NADH -> 30 ATP molecules
- makes 2 ATP per FADH -> there are 2 FADH -> 4 ATP
- net 2 ATP made in glycolysis
- 38 ATP per glucose
40
mitochondria
- outer membrane- smooth, unfolded
- inner membrane- folded (ETC is here)
- innermost part- matrix (transition rxn and krebs cycle)
- narrow space between the outer and inner membrane -> intermembrane space- plays a role in extracting energy from NADH and FADH
- phospholipid bilayer
41
anaerobic respiration
- similar to aerobic respiration (all the same stages)
- final e- acceptor is an inorganic substance other than O2
- pseudomonas aeruginosa uses nitrate ion as the final e- acceptor
- doesnt produce as much ATP
- more than 2 and less than 38
- depends on species
42
fermentation
- O2 is not used
- only glycolysis takes place
- 2 ATP are made
- organic molecule is the final e- acceptor
- not anaerobic respiration but it is anaerobic process
43
lactic acid fermentation
- only glycolysis takes place
- glucose is broken down to 2 pyruvic acid
- 2 NADH
- 2 ATP
- once pyruvic acid is made it is converted to lactic acid
- NADH is oxidized to NAD+
- pyruvic acid gets reduced to lactic acid
- regenerates NAD+
- NAD+ participates in glycolysis again to get 2 more ATP
- pyruvic acid is the organic molecule final e- acceptor
- lactobacillus does this (aerotolerant anaerobe- even in presence of O2 it doesnt use it)
44
alcohol fermentation
- glylocysis
- 2 ATP
- 2 pyruvic acid
- 2 NADH
- pyruvic acid is converted to acetaldehyde
- CO2 comes out
- NADH is oxidized to NAD+
- acetaldehyde is reduced to ethanol
- final e- acceptor is acetaldehyde
- ex. saccharomyces- yeast (Facultative anaerobe- grows in presence or absence of O2 but grows better with O2) -> that means we must make sure there is no O2 to make alcohol
- if there is O2 it will carryout aerobic respiration and make water
45
lipids
- used for energy
- when glucose isnt around
- triglyceride -> glycerol + 3 fatty acids
- exoenzyme- lipase -> breaks down triglyceride
- glycerol is then converted to dihydroxyacetone phosphate (intermediate molecules in glycolysis)
- goes into glycolysis and so on
- fatty acids that came out of triglyceride can be used too
- fatty acid is broken down into many units of acetyl CoA -> goes into krebs cycle
46
proteins
- used for energy
- when glucose isnt around
- breaks protein down into amino acids
- protein broken down by proteases
- amino acids are converted into intermediates of glycolysis or krebs cycle
47
photosynthesis
- plants and algae - chloroplasts
- chloroplasts specialize in photosynthesis
- 6CO2 + 6H2O -> C6H12O6 + 6O2
- light dependent reactions
- light independent reactions (calvin-benson reaction)
48
light dependent reaction
- chlorophyll
- cell light hits cholophyll molecules
- e- absorb light -> energized
- e- jump out of chlorophyll molecule
- e- go through electron transport chain in chloroplast
- similar to aerobic respiration ETC
- chemiosmosis -> makes ATP by photophosphorylation
- energized e- ends up with NADP+ -> NADPH
- e- that come out of chlorophyll molecule are replaced by e- from water -> breaks down water into O2 and H -> releases O2
49
DNA
- deoxyribonucleic acid
- genes
- genes are made of DNA
- DNA and genes have genetic information for structure and function of cells
- DNA is made up of many nucleotides
- nucleotides made up of: deoxyribose, phosphate, nitrogen base (differ in nitrogen base)
- nitrogen base: adenine, guanine, cytosine, thymine
- genetic information is in the nitrogen base sequence
50
structure of DNA
- double helix
- 2 chains of nucleotides
- alternating units of sugar and phosphate backbone
- nitrogen base is attached to the sugar molecule
- complementary base pairing
- adenine is not attached to phosphate its attached to sugar
- hydrogen base forms between nitrogen bases -> responsible for keeping strands together
51
complementary base pair
- cytosine pairs with guanine
- adenine pairs with thymine
- plays a major role in DNA replication and protein synthesis
52
gene
segment of DNA that codes for a functional product
- functional product- protein
- most genes code for proteins
- .1% of genes have instructions to make tRNA and rRNA
- genes are passed on from one cell to another- one generation to another
- DNA has to be replicated
53
DNA is long
- -DNA is a long molecule
- E.coli chromosomes has 4 million base pairs (nucleotides)
- DNA is replicated segment by segment bc it is so long
54
DNA replication
- replicated segment by segment
- segment unwinds and separates
- hydrogen bonds are broken
- each strand functions as a template for the synthesis of a new strand
- free DNA nucleotides are in the area of replication
- complementary base pairing takes place between the nitrogen base on free nucleotides and the nitrogen base on the template strand
- DNA polymerase links them together
- new strand spirals around the old stand
- prokaryotes- in cytoplasm
- eukaryotes- nucleus
55
replication fork
region of DNA where the replication is taking place
56
semiconservative
- an old strand and new strand
- DNA replication
- each copy of the DNA has one old and one new strand
- parent strand
- daughter strand
- survival value
- helps organism to survive under certain conditions
57
origin of replication
- where replication start
- in circular e.coli chromosome...
- made up of DNA and unique nitrogen base sequence
- 2 replication fork forms here
- one moves counterclockwise
- the other moves clockwise
- two replication forks meet -> replication is complete
- 2 copies are made
- when cell divides copies are passed onto 2 daughter cell -> identical
- genetic information has been passed form parent to daughter
58
genetic information: protein synthesis
- flows within the cell -> instruction from genes are used by the cell to make proteins
- gene is transcribed to make the mRNA
- mRNA is translated to make a protein
- transcription genetic information from the gene is coped onto mRNA
59
transcription
-make mRNA
60
translation
- genetic information from the gene is copied onto mRNA
| - make protein
61
gene
- segment of DNA
- codes for a functional product -> protein
- e. coli chromosome has 1,000s of genes
- each gene has a unique nitrogen base sequence*
- nitrogen base sequence is responsible for differentiating genes
62
promoter
where gene begins
- control regions
- unique nitrogen base sequence
- made up of DNA
63
terminator
where gene ends
- control regions
- made up of DNA
- unique nitrogen base sequence
64
coding sequence
- transcribe onto mRNA
- copied onto mRNA
- between the promoter and terminator
65
RNA polymerase
- transcription- DNA is copied onto mRNA
- makes mRNA
- enzyme
- major role in transcription
- attaches itself to the gene near the promoter
- segment of gene separates
- one strand is template strand (instructions)
- attaches RNA nucleotides together -> chain
- free RNA nucleotides are floating around where ever transcription is taking place
- once nitrogen base are exposed on template strand complementary base pairing takes place between nitrogen base on the free RNA nucleotide and the nitrogen bases on the template strand
- uracil on the free RNA pairs with the adenine on the template strand
- RNA polymerase attaches the pairs
- moves segment by segment
- polymerase is released from the gene at the terminator
- mRNA has a specific nitrogen base sequence
66
DNA polymerase
-major role in DNA replication
67
RNA
- has uracil nitrogen base instead of thymine
- single strand molecule
- made up of one chain of RNA nucleotides
- ribose sugar
68
DNA
- doesnt have uracil -> thymine instead
- deoxyribose sugar
- double strand of DNA nucleotides
69
codon
- 3 nitrogen base sequences next to each other on the mRNA
- codes for an amino acid
- different codons can code for the same amino acid -> degeneracy of the genetic code
- associated with mRNA
- ex. UAG
70
mRNA
- nitrogen base sequence of mRNA is complementary to the template strand of the gene
- form of codons
- has the genetic information in the language of RNA
- brings the message to ribosome
71
translation
-interaction between mRNA, tRNA and ribosomes
72
stop codon
- signal the end of translation
- nonsense codon
- doesnt code for an amino acid
73
degeneracy of the genetic code
- helps cell survive under certain conditions
| - different codons can code for the same amino acid
74
start codon
- translation starts
- protein synthesis starts here
- AUG
- codes for MET
75
transfer RNA (tRNA)
- one end has anticodon
- other end picks up amino acid from the cytosol
- transfers amino acids from the cytosol to the ribosome
- specific group of tRNA for each amino acid
- specificity is based on the anticodon it has
- reads the message
- ex. tRNA is specific for alanine -> cant pick up any other amino acid -> specific anticodon for alanine
76
anticodon
- triplet of nitrogen bases
| - associated with tRNA
77
ribosome
- holds mRNA so tRNA can read the message and bring the appropriate amino acid to the ribosome
- has the enzyme that attaches amino acids together (peptide bonds)
78
enzyme
- catalyzes peptide bond formation during translation
| - ribosome has the enzyme
79
translation steps
- attachment of ribosome (large and small subunit) to the mRNA near the start codon
- tRNA recognizes the codon
- tRNA brings MET to the ribosome
- complementary base pairing occurs on the codon on the mRNA and the anticodon on the tRNA
- tRNA molecules are held in place and the amino acids are next to each other
- enzyme attaches the amino acids together -> dipeptide
- dipeptide gets transferred on to tRNA and it moves on to next segment
- forms a polypeptide
- ribosome reaches stop codon -> end of translation
80
post translation
- polypeptide is released
- tRNA subunits come apart
- mRNA and tRNA is released from ribosome
- mRNA is translated again to make another copy of the polypeptide chain
81
amino acid sequence
- important for shape of protein
- important for function of protein
- based on the sequence of mRNA
- sequence of codons is based on the nitrogen base sequence of the gene from which it was transcribed
- change in nitrogen base of the gene -> change the codon of mRNA -> can change amino acid sequence
- if it is changed the protein becomes less active or inactive
82
genetic information
-flows from the gene to mRNA to protein
83
mutation
-change in the nitrogen base sequence
84
missense mutation
- single nitrogen base on a specific site on the gene is replaced by another nitrogen base
- sequence of codon is changed
- when mRNA is translated the polypeptide sequence is changed
- protein doesnt function correctly
- sometimes the mutation doesnt show up on the polypeptide chain -> codon is changed but it still codes for the same amino acid -> degeneracy of the genetic code
85
silent mutation
- sometimes the mutation doesnt show up on the polypeptide chain -> codon is changed but it still codes for the same amino acid -> degeneracy of the genetic code
- possible bc of the degeneracy of the genetic code
- survival value for the cell bc most mutations are lethal to the cell
86
cause of mutation
- can take place spontaneously
- DNA polymerase makes a mistake and inserts a wrong nitrogen base during DNA replication
- mutation frequency is increased by certain agents -> mutagens
- mutagen chemicals- nitrous acid changes shape of adenine so that it pairs with cytosine
- x-ray mutagen- pull e- out of molecules -> break in the chromosome
- UV light- mutagen
87
UV light
- mutagen
- induces the formation of thymine dimers in DNA
- adjacent thymine molecules in DNA come together
- when DNA is replicated DNA polymerase is confused
- inserts the wrong nitrogen base in the new DNA being synthesized
- cells have evolved so that enzymes can separate thymine dimers
- if there are too many thymine dimers not all the thymine dimers will be separated -> accumulate -> mutation
- sunlight
- accumulation of thymine dimers causes mutations in skin cells -> skin cancer
- caused by excessive sun tanning
88
genetic transfer and recombination
- contributes to genetic diversity in a bacterial population
- new strains pop up -> genetic recombination is partly responsible
- 2 DNA are in the same cell and come in contact -> pieces of DNA are exchanged
- each DNA molecule becomes a recombinant DNA
89
crossing over
- chromosome A and chromosome B in the same cell come in contact
- random process
- twist around each other
- pieces of DNA are exchanged
- makes recombinant chromosome
90
genetic transfer
- 2 DNA in the same cell
- piece of DNA is transferred from a donor to a recipient
- bacteria has one DNA molecule
- if genetic transfer takes place the bacteria can have 2 DNA molecules
- 3 methods of transfer:
- transformation
- conjugation
- transduction
91
genetic transfer: transformation
- DNA from a donor cell is transferred to recipient
- donor cell is dead
- when bacterial cell dies the DNA is released into the environment
- DNA gets fragmented into pieces
- recipient cell comes in contact
- DNA penetrates cell wall of recipient -> 2 DNA molecules
- own chromosome and donor DNA present
- when the own chromosome and donor DNA come in contact -> crossing over
- donor DNA aligns with complementary bases
- *can make the recipient cell more pathogenic -> picks up genes that can code for capsules
- becomes a capsulated bacteria- more pathogenic bc capsule protects bacteria from phagocytosis
92
genetic transfer: conjugation
- subspecies of the same cell
- F+- has the pilus (filamentous structure found on the surface) and small circular DNA (F plasmid/factor)
- F+ cell has plasmid and chromosome (they are separate)
- F-- does not have pilus
- F+ uses it pilus and attached to F- and conjugates
- F plasmid- has genes for the pilus
- plasmid gets replicated and copy gets transferred to the F- cell through the pilus
- F- becomes F+ -> makes two F+ cells
- F+ has 2 DNA molecules (chromosome and plasmid)
- f plasmid gets inserted into chromosome -> becomes an Hfr cell (high frequency of recombination cell)
- Hfr cell- very good at conjugation
- Hfr cell- makes the pilus
- Hfr and F- cell conjugation:
- during conjugation the DNA gets replicated and starts in the middle of the f plasmid
- piece of f plasmid and piece of chromosome get replicated and transferred into the F- cell
- F- cell never gets the entire chromosome or plasmid bc it is much larger than the cell and they dont stay conjugated for long enough
- F- gets only a piece of donor DNA and plasmid -> inserts into chromosome and becomes recombinant -> doesnt become F+ cell and does not make pilus
- can make an F- cell resistant after it picks up DNA from another Hfr cell -> shares resistance
93
genetic transfer: transduction
- DNA of donor cell is transferred with recipient cell
- bacteriophage is a virus (acellular) that infects bacteria
- bacteriophage picks up donor DNA and releases it into recipient cell
- bacteriophage gets into host cell to reproduce itself
- bacteriophage attached to donor cell
- phage DNA gets released into host
- phage DNA gets replicated
- donor chromosome gets fragmented
- assembly of phage takes place ->
- by mistake sometimes fragments of bacterial DNA gets enclosed into the protein code of the phage
- transducing phages- have bacterial DNA in them instead of phage DNA
- donor cell breaks down and dies
- phages are released including transducing phages
- transducing phage comes in contact with bacteria and releases donor DNA into bacteria (receiving cell)
- donor DNA gets inserted into the chromosome of the recipient cell -> recombinant
- sometimes transducing phages pick up toxic genes and spreads it
94
regulation of gene expression
- most genes are expressed constantly
- constitutive genes- constantly transcribed and translated and expressed
- genes that code for enzymes of glycolysis are constitutive genes
- hexokinase gene is a constitutive gene
- some genes are expressed only when their products are needed -> inducible genes
- beta galactosidase gene is an inducible genes
95
beta galactosidase gene
- inducible gene
- codes for the enzyme beta galactosidase
- enzyme breaks down lactose to glucose and galactose
- needed only when lactose is in the medium
- expressed in the presence of lactose
- gene is part of the lactose operon
- lactose operon is located on e. coli chromosome
96
operon
- many genes are controlled by the same control region (promoter)
- has many genes
- controlled by the same control region (promoter)
- regulation
- genes of the same operon share a promoter
97
lactose operon
- 3 structural genes
- Z- beta galactosidase
- Y- permease- transports lactose into cytoplasm
- A- transacetylase
- controlled by the same promoter and operator
- each gene has its own nitrogen base sequence
- next to lactose operon -> I gene- regulatory gene - regulates the expression of lactose operon -> codes for repressor protein
- in the absence of lactose the lactose operon is inactive
98
regulation of lactose operon: inactive lactose operon
- repressor protein hop onto the operator protein and block RNA polymerase
- when RNA polymerase attached to promoter it cannot get to structural genes bc of the repressor blockage
- only when the RNA polymerase is able to pass over the structural genes will the mRNA of the structural genes will be made
- no mRNA -> no translation -> no proteins
- inactivates lactose operon
99
regulation of lactose operon: active lactose operon
- if lactose is in environment it will bind to repressor protein -> inactive repressor protein
- pulls the repressor protein from the lactose operator -> no more blockage
- RNA polymerase is able to make mRNA for the structural genes
- lactose activates the lactose operator by inactivating the suppressor
100
repressor protein
- on the operator
- if something is bound it is pulled form the operator -> inactive
- if nothing is bound it block RNA polymerase from making mRNA of the structural genes
101
regulation of lactose operon: in presence of glucose and lactose
- carabolite repression
- if glucose and lactose are present
- cell will use glucose over lactose
- doesnt need beta galactose
- RNA polymerase has a hard time attaching the promoter
- glucose prevents the RNA polymerase from attaching
- prevents mRNA coding
- once glucose is used up the RNA polymerase can easily attach to promoter and make mRNA
- lactose binds to suppressor and actives lactose operon
- as long as glucose is present lactose operon is inactive
102
catabolite repression
-in the presence of glucose catabolism of other sugars is repressed
103
lactose operon
- active in absence of glucose and presence of lactose
| - both conditions have to be satisfied for the activation of the lactose operon
104
growth of cells that have glucose only vs lactose only
- cells grow fast with glucose only
- cells grow slower with lactose only
- lactose is a disaccharide -> needs to break down lactose first -> time consuming
105
growth of cell that have glucose and lactose
- uses all the glucose up first (fast rate)
- lag time- cell stops growing for a while after glucose runs out
- cell starts growing again by using lactose (slower)
- cells stop growing for a bit bc it takes a while for the cells to activate the lactose operon
106
inducible gene
- beta galactosidase gene
- helps the cell to save its energy and chemical resources such as amino acids
- cell is not making something that it does not need
107
plasmids (R plasmid)
- small circular DNA
- R plasmids- resistance plasmids
- R plasmid genes code for antibiotic resistance
- they code for enzymes that break down antibiotics
- bacteria is not killed by antibiotics
- unique to prokaryotic cells
108
R- bacterial cell
- does not have r-plasmid
- sensitive to antibiotics
- damage to cell wall
109
R+ bacterial cell
- has r-plasmid
- resistant to antibiotics
- r-plasmid has a gene that codes for penicillinase
- enzyme breaks down antibiotic
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R100 plasmid
- resistance for many different antibiotics
- also has genes for making pilus
- if bacteria picks up this plasmid it will be very resistant
- pilus allows conjugation and transfer of plasmid to other bacteria
- can be transferred between E. coli, klebsiella, and salmonella
- resistance spreads
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dissimilation plasmids
- have genes that code for enzymes that break down petroleum
- found in pseudomonas
- used in bioremediation- use of microbes to clean up chemical pollutants
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bacteriocin plasmids
- code for toxins
- toxic to certain species of bacteria
- ex. lactococcus lactis has a bacteriocin plasmid
- codes for toxin -> nisin
- nisin prevents the germination of clostridium endospore
- helps lactococcus lactis -> prevents growth of other bacteria so it has more nutrients for itself
- preserve cheese
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transposons
- small segment of DNA
- transposed (move) one region of DNA to another
- jumping genes
- can cause problems by messing up sequences
- doesnt move often tho
- found in all organisms
- simple transposons (insertion sequences)- has a gene that codes for an enzyme -> transposase
- transposase- helps simple transposon to move from one part of the DNA to another
- unique nitrogen base sequence on each side
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transposition
-cutting and resealing of DNA
115
complex transposons
- unique nitrogen base sequence on each side
- made up of 2 insertion sequences
- in between there is a gene that codes for antibiotic resistance
- found in bacteria
- often found in r-plasmid
- can move from the plasmid to the chromosome
- can be transferred through conjugation
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genetic engineering
- deliberate manipulation of genes in an organism
- done in a lab by scientists
- makes therapeutic substances such as human insulin
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insulin
- came from pancreas obtained from slaughtered animals before genetic engineering -> did work well
- genetically engineered E. coli cells to make human insulin
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genetic engineering mechanisms
- use plasmid (small circular DNA found in some bacterial cells) as a vector and insert a gene of interest (human insulin) into the plasmid
- plasmid becomes a recombinant plasmid
- recombinant plasmid is introduced into a bacterial cell (E. coli) -> becomes the recombinant cell/transformed bacteria
- E. coli transcribes and translates the gene and makes the human insulin
- once recombinant cell is made it can be grown in a nutrient broth like any other e. coli cell -> descendants of the recombinant will also be recombinant
- easy to make a lot human insulin
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tools used in genetic engineering: restriction enzymes
- restriction enzymes come from bacteria
- used to breakdown phage DNA in the bacteria
- extract the restriction enzyme from bacteria and use it for genetic engineering
- EcoR1, BamHI -> recognize specific sequences
- restriction enzymes make staggered cuts in the DNA
- they fragment DNA
- ends of the fragment are single stranded
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Restriction enzyme: EcoR1 example
- restriction enzyme cuts double stranded DNA at he specific nitrogen base sequence it recognizes (can have more than one site of recognition)
- staggered cuts DNA and produces a fragment with 2 sticky ends
- bc the cuts are staggered a piece of DNA comes out and has single stranded ends -> sticky/cohesive ends
- restriction enzyme cuts a piece of bacteria DNA and human DNA -> bc they are cut at the same recognition sites they have the same nitrogen base sequence on the sticky ends
- this allows the piece of DNA to join by complementary base pairing -> recombinant DNA
- enzyme DNA ligase is used to unite the two DNA fragments
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vectors
- carry the gene of interest into a bacterial cell
- plasmids
- small enough -> they can enter into the cell easily
- they have selection markers which are antibiotic resistance genes
- antibiotic resistant genes are on the plasmid and help in selecting the cells that have the gene of interest
- acts like a tag
- plasmid has recognition sites that restriction enzymes can recognize
- has selection markers (antibiotic resistance genes)-> amp
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introducing the recombinant plasmid into the cell
- take a bunch of recombinants and put them into a tube with host cell (e. coli)
- some of the e. coli will come in contact with the recombinant plasmid and pick it up and some will not
- incubation
- there will be two populations of e.coli (one with recombinant and one without)
- they select the recombinant cell by plating the mixture on the medium with the ampicillin antibiotic -> incubate
- the colonies that show up on the plate are the ones with the recombinant plasmids bc they have the selection markers (antibiotic resistant genes) in their plasmid
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cDNA
- complementary DNA
- cDNA does not exist in nature
- cDNA- synthetic gene that only has exons
- mRNA is used to make cDNA (by scientists- not in nature)
- DNA nucleotides and reverse transcriptase enzymes are added to the tube with mRNA
- reverse transcriptase uses mRNA as a template to make a complementary strand of DNA
- DNA polymerase is then added to the tube and uses the DNA strand as a sample to make the second strand -> makes cDNA
- cDNA only has exons
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eukaryotic genes: introns and exons
- introns- noncoding regions
- exons- coding regions
- prokaryotic genes only gave exons
- when the eukaryotic gene is transcribed the RNA also has exons and introns
- eukaryotic cells have certain enzymes that remove introns and stitch the exons to make the mRNA
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why do we make cDNA
- if we want to introduce eukaryotic gene into a prokaryotic cell we use cDNA
- if we place natural eukaryotic gene into bacterial cell it wont be able to remove the introns
- functional protein will not be produced by the prokaryotic cell
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applications of genetic engineering
- used to make therapeutic substances (insulin make by e. coli)
- hormones- insulin
- used to make growth hormone -> somatotropin (treats stunted growth)
- produced by genetically engineered E. Coli cells
- before genetic engineering they got growth hormone from pituitary gland removed during autopsy -> transferred diseases
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vaccine: hepatitis B vaccine
- genetic engineering is used to make vaccines
- hepatitis B vaccine:
- genetically engineered saccharomyces cells (eukaryotic)
- vaccine has only the protein part of the virus
- does not have the genetic material of the virus
- no chance of getting the disease due to vaccination
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E. coli
-prokaryote
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genetic screening
- DNA technology is used
- used to see if someone is a genetic carrier for a genetic disorder
- xeroderma pigmentosum (XP)- genetic disorder
- there is a mutation in a gene-> the gene codes for an enzyme that repairs DNA damage caused by UV rays
- enzyme is not functional
- sensitive to sunlight -> get sunburn within a few minutes
- various health problems are also associated with this condition
- if a person has the diesase -> there are symptoms
- some people can be carriers -> one XP gene (father) one normal gene (mother) -> heterozygous for XP (no symptoms)
- if 2 carriers have a baby the baby can have the disease
- uses the southern blotting procedure
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southern blotting
- used in genetic screening
- take blood and DNA
- fragment DNA using restriction enzyme (made from bacteria)
- DNA fragments are separated by gel electrophoresis by size
- the DNA bands show up
- smaller pieces move faster in the gel (more further down the smaller)
- DNA bands are transferred onto nitrocellulose filter/membrane
- nitrocellulose filter holds onto molecules like DNA
- place the nitrocellulose filter and attached DNA into a zip lock bag
- a solution containing many copies of the radioactively labeled probe (complementary to gene of interest) is added
- incubate
- probe will hybridize/comes together with gene of interest through complementary base pairing
- remove and rinse nitrocellulose filter
- expose to x-ray film
- where ever there there is radioactive activity it will blacken -> tells us where the probe and therefore the gene of interest is
- a black band shows up for carrier
- a black band show up larger and wider for someone with the disease (bc twice as many copies of the gene of interest)
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probe
-single strand of DNA that is complementary to the DNA of interest
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genetic engineering: agriculture
- makes plants that are resistant to insects
- take BT toxin gene from bacillus thuringiensis
- put the BT toxin gene into a vector (plasmid) -> recombinant plasmid
- recombinant plasmid is introduced into pseudomonas fluorescens bacteria
- transformation process is used to transfer DNA -> bacteria picks up DNA from its environment
- recombinant pseudomonas fluorescens cell makes BT toxin
- pseudomonas fluorescens is released onto the leaves of the plant -> reproduce other recombinant cells -> covers the leaves with BT toxins
- when insect nibbles on leaves it ingest toxins and dies
- the plant itself is not genetically engineered -> it has genetically engineered bacteria on it
- we cant just use bacillus thuringiensis bc it cannot colonize the leaf -> they only live in soil
- as long as the plant is alive it will be resistant
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PCR
- polymerase chain reaction
- used to amplify DNA
- makes more DNA to get more sample for testing
- target DNA is placed in a tube with a solution, DNA nucleotides, and DNA polymerase
- tube is heated and cooled several times -> causes the DNA to become replicated
- billions of copies of target DNA are made at the end of PCR
Microbiology
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