Paper 3 Flashcards
definition of biotech
use of a biological organism to generate a product for medical, industrial or agricultural use
three basic requirements of patentability
novelty, non-obviousness, industrial utility
lifetime of a patent
around 20 years
cost of goods
accumulated total of all costs used to create a product or service
what is humulin?
a recombinant human insulin protein
which product dominates pharm?
monclonal antibodies, followed by hormones, clotting factors, enzymes, vaccines etc
where are mAbs usually produced?
CHOs
early mAbs were produced in hybridoma cell lines
where is the current bottleneck for mAb production?
downstream purification
what is gene therapy technology
replacing/augmenting defective genes with normal copies of the gene
old adage
prevention better than cure
covid prevention, detection and treatment
- vaccine
- LFT, PCR, LAMP, sequencing
- mAbs, antivirals, dexamethazone - repurposing old drugs - normally used to treat comatose ppl
What did Oró discover in 1961
NAs can form spontaneously from HCN in water-ammonia systems under the conditions that are assumed to have existed on the primitive Earth
what has been suggested as the birthplace of life?
deep sea hydrothermal vents
was the primitive environment on Earth oxidising or reducing?
reducing
conservative mutation
letter changes but no change in function
which gene is used as the standard for classification and identification of microbes?
16S rRNA
what three main groups are cells classified into?
bacteria, archaea, eukaryotes
what is LUCA and LECA
last universal common ancestor, last eukaryotic common ancestor
when does a cell duplicate its entire genome?
when it divides into two daughter cells
ortholog, paralog and homolog
genes in two separate species from the same ancestral gene in the last common ancestor
- related genes resulting from gene duplication within a single genome
- genes related by descent in either way
properties of eukaryotic cells
DNA in nucleus, larger, have a cytoskeleton, don’t have a tough cell wall, have mitochondria unlike prokaryotes
endosymbiont hypothesis
early anaerobic cell engulfed an aerobic prokaryotic cell - new cell can live anaerobically and aerobically
could mitochondria have come from a purple photosynthetic bacterium?
maybe - mitochondria have similar size to bacteria, have their own DNA and ribosomes, have a double membrane
organotrophic
can use anything as food - sugars, amino acids, hydrocarbons, methane etc. e.g. E coli
are yeast unicellular or multicellular?
unicellular eukaryotic cells
how can yeast reproduce
vegetatively (by simple cell division) or sexually
a similarity btw chloroplasts and mitochondria
contain their own genome
important difference btw mitochondria and chloroplasts
cplasts: inner membrane not folded into cristae and doesn’t contain electron transport chains
instead the thylakoid membrane contains ETC, photosynthetic light harvesting systems and ATP synthase
E coli
gram negative, facultative anaerobic, rod-shaped, coliform bacterium found in the lower intestine of endotherms
20 minute reproduction time
what do most antibiotics inhibit?
either bacterial protein synthesis
or bacterial cell wall synthesis
which genus are most antibiotics made of
Gram-positive genus Streptomyces
what do penicillins target?
cell wall synthesis
cross linking of peptides on the polysaccharide chains is prevented
penicillin is a suicide substrate
three main ways by which a pathogen can develop drug resistance
- alter the molecular target of the drug so that it is no longer sensitive to the drug
- produce an enzyme that destroys the drug
- prevent access to the target by e.g. actively pumping the drug out of the pathogen
‘MOTEP’
penicillinases
aka beta-lactamases
enzymes produced by structurally susceptible bacteria which render penicillin useless by hydrolysing the peptide bond in the beta-lactam ring of the nucleus
if cells have approximately the same DNA why do they behave differently?
different genes are expressed in different cells and at different times
what is special about the 5’ to 3’ direction?
it is the direction in which DNA is synthesised
what is cool about RNA?
can catalyses so may be the original biomolecule
what does a parental DNA helix do?
produce identical daughter DNA helices
how many nucleotide pairs in the human genome?
3.2 bn
what rxn does the enzyme DNA polymerase catalyse?
addition of a deoxyribonucleotide tot he 3’ end of a polynucleotide
4 levels of protein structure
1 amino acids
2 folding e.g. alpha helix/beta sheet
3 how it folds in 3D
4 multiple domains
three mechanisms of proof-reading
DNA, exonucleolytic and mismatch repair
transcription
- unwind small portion of DNA double helix to expose bases on each DNA strand
- one of the two strands of the DNA double helix then acts as a template for the synthesis of an RNA molecule
- nucleotide sequence of the RNA chain determined by the complementary base pairing btw incoming nucleotides and the DNA template
- RNA polymerase is responsible for unwinding the DNA helix just ahead of the active site for polymerisation.
- growing RNA chain extended one nucleotide at a time in the 5’ to 3’ direction
UNTECOPO 5
how many transcripts per hour can be made from a single gene?
over a thousand
do RNA polymerases need to be as accurate as DNA polymerases?
no, bc don’t PERMANENTLY store genetic info in cells
differences in transcription btw eukaryotes and prokaryotes
bacteria 1 type of RNA polymerase, eukaryotes 3
bacterial RNA polymerase only needs one sigma factor, euk need many transcription factors
initiation harder for euk
in euks, RNA molec resulting from transcription has both INTRONS AND EXONS
alternative splicing
different introns can be removed for different gene products, or exons can be of different lengths
for translation, in which direction is the codon read?
5’ to 3’ direction
what do aminoacyl tRNA synthetases do?
they have a two step mechanism which ensures that the correct amino acid is bound to the correct tRNA molecule
how many binding sites does a ribosome have for tRNA?
3: E P A
exit, peptidyl, aminoacyl
three main steps in translation
initiation, elongation and termination
why has the ribosome been a frequent target of antibiotic molecs?
prok and euk ribosomes quite different, w/o ribosome protein synthesis is screwed
how does covid replicate?
has RNA and needs to make lots of copies - does that using a polymerase. Uses error prone replication - not concerned with high fidelity copying bc will have an evolutionary advantage if it mutates a lot. changes in spike protein mean undetectable. proof-reading leads to cumulative mutations. delta to omicron 100 mutations
lac operon
a polycistronic transcript which can make multiple proteins from one mRNA transcript
what can incorrectly folded proteins lead to?
neurodegenerative diseases e.g. Alzheimer’s or Parkinson’s
are lipid bilayers thicker for saturated or unsaturated lipids?
saturated lipids
why are exosomes an interesting therapeutic product?
diagnostics, precision medicine
can easily cross cell barriers so could deliver NA based therapies
what does covid do?
- binds 2. fuses with cell membrane 3. uncoats and NA released. 4. NA processed by viral proteases 5. Viral RNA which can code for 9 to 11 proteins is synthesised 6. Virus encapsulated 7. Virus buds from vesicles 8. Virus emerges from cell membrane
bifunc rena pro cabudem
what are the opportunities for developing antivirals
specifically target RNA genome replication bc virus has an RNA polymerase specific to itself
is inside or outside of a cell more positive
outside of a cell
what are the two major classes of membrane transport proteins?
transporters and channels
sth that is amphiphillic
transmembrane protein
what is the equilibrium potential for an ion?
voltage gradient (membrane potential) at which equilibrium is reached. It is given by V in the Nernst equation at eq /\Gconc+/\Gvolt = 0
what does the transporter GLUT4 do?
allows glucose to pass from cells to blood via simple diffusion. Type II diabetes mellitus - cells don’t insert GLUT4 into their membranes so high BGC so heart disease/stroke/kidney failure
why does ATP => ADP + Pi release energy?
release of terminal phosphate removes an unfavourable repulsion btw adjacent negative charges. When released, the inorganic phosphate is stabilised by resonance and by favourable bond formation with water.
is NADH an intermediate in anabolic or catabolic rxns
catabolic
what is glycolysis?
glucose to two pyruvates, releasing two ATPs and 2 NADHs
why are reactive oxygen species contained within mitochondria?
oxygen is toxic
what does PCR do?
enable amplification of the number of DNA copies
when did recombinant DNA tech really take off?
In 1978 when Genentech made synthetic human insulin
why do we need the Taq polymerase for PCR?
PCR based on thermocycle. Need 90 deg to separate two DNA strands, but this destroys DNA polymerase which likes 37 deg C. So need a heat resistant DNA polymerase, i.e. Thermophilus aquaticus.
is DNA charged?
Yes, negatively
what does bla do?
codes for amphicillin resistance
lac repressor
catabolyte repression to maximise bacterial growth
what is good about yeast expression vectors compared to plasmids?
can be used to express euk proteins needing post-translational modifications
also allows cloning of v large DNA molecs
cDNA
for cloning eukaryotic genes
cDNA produced from fully transcribed mRNA found in the nucleus and therefore only contains the EXPRESSED genes of an organism
protein purification methods
three main types of column chromatography: ion-exchange, gel-filtration, affinity
isoelectric point
pH at which a protein has no net charge
if pH > pI, protein has net positive charge
what dye is used for SDS PAGE?
Coomassie blue
how can you kill a tumour without using chemotherapy?
use the patient’s OWN T cells in treatment to minimise chance of rejection. Genetically modify them to form a chimera (mixture of two things). So get specific killing of tumour, no need for chemotherapy
what are clones in the context of cells?
cells derived from a common ancestor cell
type I diabetes
immune rxns against insulin-secreting cells in the pancreas kill these cells, leading to severe insulin deficiency, high BGC and body wasting
i.e. beta cells destroyed by autoimmune rxn
what is the major class of immunoglobulin in the blood?
IgG
polyclonal antibodies
cheap, rapid, large quantities of non specific anitbodies, recognise multiple epitopes on an antigen, different batches vary in composition
uses of enzymes
Textiles: catalase to remove H2O2
Food and drink: Fungal alpha amylase for dough improvement
Pulp: Cellulase for pulp refining
specific activity
number of units of enzyme activity per mg of total protein present (including impurities)
what does lysozyme do?
destroys bacterial cell walls by HYDROLYSING the sugar linkages between NAM and NAG in peptidoglycan.
Glu 35 and Asp 52 are the catalytic residues in lysozyme
substrate is an oligosaccharide of six sugars.
final products are an oligosaccharide of four sugars and a disaccharide, produced by hydrolysis
which vitamin is the precursor of the coenzyme NAD+?
niacin
issues with Lineweaver Burk plot
need a lot of data points at low substrate concentration
what can lead to enzyme inactivity?
active site blockage, inactivation of catalytic groups, change in the shape of the active site
examples of post-translational modifications
methylation, acetylation, glycosylation
effect of immobilisation on rmax
rmax decreases
which gene regulatory protein is regularly mutated in cancers?
p53 - for detecting DNA damage in cell cycle control and arresting the cycle
meiosis
- meiotic S phase
then two rounds of chromosome segregation
meiosis I segregates the homologs
meiosis II segregates the sister chromatids
mitosis vs meiosis
mitosis: homologs don’t pair up and the sister chromatids segregate in a single division
insulin production
insulin is a peptide hormone of 51 amino acids (A chain and B chain which are linked by disulfide bonds). Synthesised as a larger protein (proinsulin) then cleaved by proteolytic enzymes. PTM of proinsulin to insulin only occurs in the beta cells of the islets of Langerhans
RQ
CO2 made/O2 used in mol/s for both
what does a high degree of reduction imply?
a low degree of oxidation
oxygen utilisation rate
O2 used/S used in mol/s for both
when are secondary metabolites starting to be produced?
in the stationary phase, e.g. penicillin
vs e.g. alcohol as a primary metabolite in the growth phase
what type of rxn is cell growth?
autocatalytic - catalyst is product of rxn
generation time
time required to double the cell concentration
maintenance energy
substrate consumed for non-growth functions
e.g. repair, osmotic balance, motility
essential for homeostasis
directly growth associated
related to energy generation, rate of product formation prop to growth rate of cells
growth associated products formed as a result of the primary metabolic functions of the cells
e.g. ethanol production by the anaerobic fermentation of glucose by yeast
what phase do cells need to be in for a chemostat to work?
exponential growth phase
how do adaptor molecs enable ribosomes to translate mRNA sequences into protein sequences?
The adaptor molecs are tRNAs. These short RNA molecs have an anti-codon which is a 3-base nucleotide sequence which complements and base pairs with a specific codon in the mRNA. so each codon (apart from the three stop codons) needs a complementary tRNA. the corresponding amino acid is covalently linked to the other end of the tRNA molec by a specific enzyme (an amino acyl tRNA synthetase) to form an amino acyl (or charged) tRNA. Ribosomes move along the mRNA reading one codon at a time and a specific amino acyl tRNA binds to the ribosome according to which codon is being read.
difference between synthetic biology and genetic engineering
synthetic bio involves many more genes
synthesis of poliovirus genome from nucleotide monomers (7741 bases)
what about a CSTR is better than a PFR?
PFR: poorer T ctrl, undesired thermal gradients and shutdown cleaning more difficult and dear. To deal with exothermic nature of a rxn, CSTR cd have a cooling jacket
