DNA to study protein function Flashcards
Why is it hard to determine/sequence ancient DNA?
What are 4 advantages to expressing recombinant proteins in mammalian cells?
What are recombinant proteins used for?
What are the disadvantages of using mammalian cells as hosts?
DNA degrades overtime, bases deaminate
- correct protein folding (environment)
- post translational modifications (bacteria don’t glycosylate)
- sub-cellular protein targeting
- biological activity present
research, medicine, industry (diagnostics)
- expensive
- not easy to produce large yields
How can you overcome the small yield problem producing recombinant proteins in mammalian cells?
What is crucial for the shuttle vector?
What enzyme is required for the cloned DNA gene in mammals?
How does the mammalian selection system work?
How can you induce transfection of shuttle vector into mammalian cell?
What is the difference between transient & stable transfection?
Cloning vector:shuttle vectors - clone in bacteria & express in mammals
Mammalian promoter, polyA signal for mRNA to have polyA tail (stability of mRNA), 2nd mammalian promotor with neo R gene (selection), antibiotic selection for bacteria, ori for both species
RNA polymerase II
NeoR gene counteracts chemical G418 - so a transfected mammalian cell will die if don’t contain the shuttle vector/integrated DNA
Lipid treatment - coats DNA & allow fuse with cell membrane
Electroporation - holes in cell membrane
Transient - plasmid survives 48-72h
Stable - DNA integrates into host chromosome
Why are insect cells more advantageous than mammalian cells and bacteria? 5
What are the 2 downsides?
What cloning/donor vector is used in insects? What are the cons?
What system in the virus is used for protein expression?
What features are on the donor plasmid with cloned DNA? 2
How is the plasmid inserted into the baculovirus bacmid (genome)?
What are the steps? Briefly 5
more robust
cheaper
correct protein folding - higher eukaryotic environment
subcellular targetting
post trans modification
glycosylation slightly different in insects than mammals
cloning is difficult
Baculovirus cloning vector/plasmid- larger genome, difficult to manipulate & fragile
bac-to-bac
Polyhedrin promoter so DNA can be transcribed in insect cells
Transposition sequences to transpose DNA from baculovirus cloning/donor vector into bacmid genome (e.coli)
Helper plasmid with virulence mechanism helps donor plasmid insert into bacmid using transposition
Cloning donor plasmid -> inserted into bacterial bacmid genome -> grow & isolate bacmid -> infect insects with bacmid -> infect insects with baculovirus product
How are the recombinant bacmids selected in e.coli?
What happens after with the recombinant bacmid?
What happens following transcription & translation in the insects?
how can you use this product?
How do you make recombinant proteins from this?
lac z gene (beta galactosidase) so on insertion the gene is displaced & produce white colonies
Isolate the bacmid DNA & infect insect cells with it
Baculovirus are produced
To have large supply of baculovirus so won’t have to keep making recombinant bacmids & reinfecting
Infect the insects with baculovirus & they hijack cell machinery to produce
What have recombinant proteins be used for in protein therapy?
What are its limitations?
Why are recombinant proteins better than natural ones?
Recombinant hormones, blood clotting factors, vaccines
Very expensive, delivery is difficult
fewer off-target effects/side effects
What are the 4 uses for oligonucleotide-based site-directed mutagenesis?
What are the 4 requirements?
In what state must plasmid be containing the cloned DNA?
How does this allow the first step?
What primers are added if wanting to change template CCC to CAC? In both strands (GGG on other)
What happens after the primers are added?
What polymerase is added and why?
What happens after strand synthesis?
What are the 4 different products possible?
identify key amino acid residues, make protein more stable, create new functions of a protein, identify how mutations cause disease
Cloned DNA (template), DNA polymerase, dNTPs, primer (with mutation)
Methylated
- Dpn1 enzyme cleaves methylated DNA (GA>TC) sites
- GTG primer added to CCC template and CAC primer added to GGG template
- Denature the 2 strands of DNA & then allow primed synthesis of new strands with mutagenic primers with DNA polymerase and dNTPs
Pfu - proof-reading activity
- Heat plasmids to allow re-annealing of all possible DNA strands
2 x new strands - neither methylated (want)
2 x parent strands - both methylated
2 x (1 x new strand and 1 x parent strand) = hemi-methylated = hybrids
Following formation of 4 plasmids in site-directed mutagenesis, dpn1 is added. What does this do?
What is the end result?
What happens following this selection?
What are the general applications of site-directed mutagenesis?
Digested methylated/hemi-methylated DNA
Only newly synthesised plasmid stays as has both mutations in both strands
Bacterial cells transformed with mutated plasmid for replication
Alter protein sequence with point mutation/insertion/deletion - primer design
Deletion - shorter primer
How is the fluorophore in GFP made?
What are the problems with GFP?
How is this solved?
Where is a GFP protein added to cloning DNA?
Why?
How did GFP show that GLUT4 cells moved location in fat cells with and without insulin?
Cyclisation & oxidation of Ser65, Tyr66 & Gly67 tripeptide
Isolated in jellyfish at 28C - mammalian cells are 37
Site-directed mutagenesis - fluoresce at 37C & introduce new colours with emission variants
C-terminus
N-terminus can affect protein localisation
Without insulin - protein accumulates inside cell
With insulin - protein accumulates to surface - fluorescence
What is the FMO gene family?
What reaction does FMO3 catalyse?
What is pungent odour caused by?
What was the change in the FMO3 gene that showed the codon change in patients?
How did they correlate FMO3 activity with odour? What was the conclusion?
What was the difference in translating FMO2 mRNA wild-type & mutant?
What happens with either protein & its response to the anti-TB drug ethionamide?
Flavin-containing monooxygenase
Trimethylamine intro trimethylamine N-oxide in the liver to be excreted in kidneys
Excretion of trimethylamine
CCC to CTC (patient)
Site-directed mutagenesis of CCC to CTC in FMO3 & expressed both of these in insect cells & measured the enzyme activity. FMO3 CCC has enzyme activity whereas FMO3 CTC didn’t
Wild-type produced a longer protein than the mutant (pre-mature stop codon - non-functional)
Drug enters bacterium & converted into sulfenic acid by long protein
Short protein uses drug & converts into sulfenic acid which taken up by host cells in lungs causing damage
Where is FMO2 & FMO3 expressed?
How was the phenotype of mice observed with and without FMO5?
What was observed in older mice with and without the gene?
What else differed in the microbiome of WT and KO mice?
How were these bacteria identified?
What is FMO5 responsible for in animals?
FMO2 - lungs
FMO3 - liver
FMO5 gene knocked out with cloning
Wild type mice gained lots of weight & lots of fat in cells, whereas knocked out mice didn’t store fat but had same number of cells
KO mice had more bacteriodetes than firmicutes than wild type
Sequencing 16S rRNA of bacteria in gut & comparing with databases of known 16S rRNA
Make microbial molecule that influences fat deposits & plasma cholesterol
