methods 7 Flashcards
how are protein affinity purification, pulldowns, and co-immunoprecipitation similar and different?
similar: methods to get protein out of a complex mixture using affinity of protein to a ligand/antibody
differences:
affinity purification uses an over expressed protein from a bacteria (so you may also purify bacterial interaction proteins), while pulldowns and co-ips use native cells.
co-ip proteins do not have a tag, while affinity purification and pulldowns downs do
co-ip is done with live cells that you fix in place to see what interactions naturally occur
the tagged bait protein in pulldowns in recombinant, while prey is not (or vice versa)
What is the bait and what is the prey in yeast 3 hybrid?
it’s arbitrary, you can either use known RNA as the bait to grab a protein, or known protein to grab an RNA (you would still have make a hybrid RNA/hybrid protein so you need to know the potential interacting partners)
why is it more common to use RNA as the bait in a yeast two hybrid?
there are fewer potential protein interaction partners you need to subclone hybrids for compared to the potential RNA interactors you would have to subclone hybrids for
throughput of yeast two hybrid?
Low* to high
- One bait protein can be tested against a library of preys
throughput of pull-down assay
low to medium
throughput of co-immunoprecipitation assay
low to medium
throughput of FRET
low*
- One bait protein can be tested against a library of preys
throughput of bimolecular fluorecence complementation (BiFC) or split YFP
low*
- One bait protein can be tested against a library of preys
what is RNAse I footprinting use to measure?
RNA protein interactions
How does RNAse I footprinting work?
RNA is end labeled, incubated with protein of interest, fragemented with RNase I, resolved on gel and radiographed
What does RNA immunoprecipitation measure
RNA-protein interactions
how does RNA immunoprecipitation (RIP) work
RNA-protein complexes in lysate are incubated with protein beads (have an antibody against protein of interest or ligand if protein was tagged), beads isolated, RNA extracted, adaptor ligation, cDNA synthesis, PCR, next gen sequencing performed to id interaction partners
how to measure weak protein-RNA interactions with RIP?
crosslink the RNA/protein complexes with UV before performing RNA immunoprecipitation
What does a functional protein array measure/how?
A chip is covered in tiny droplets of recombinant proteins, screen interaction with a variety of labeled molecules (RNA, DNA, proteins, carbs, lipid, antibodies, etc) to detect interactions
commonalities of genome editing tools
recognize target sequence
cut dsDNA in pre-designated location
trigger dsDNA repair machinery to either: re-join cut ends with NHEJ (small deletions/insertions) or HR (insert template DNA)
what is a nuclease
an enzyme that cleaves nucleotides
how many nucleotides does one zinc finger recognize?
3
what happens when you attach a nuclease to a zinc finger
you have a zinc finger nuclease able to recognize a specific 3 bp sequence and cut dsDNA
name of common nuclease combined with zinc fingers and TALENs
Fok1
limitations of zinc finger nucleases
difficult to engineer
limited number of available zinc fingers
spacing the assemble correctly is difficult
what does TALEN stand for
transcription activator-like effector nuclease
components of a TALEN
Fok1 domain fused to a customizable DNA-binding domain
What is a TAL effector (TALE) and how does it work
a protein able to bind DNA, recognizes DNA sequences by unique amino acid code in the middle of the protein. Variations in the amino acid code determine what bps it will bind
what does CRISPR stand for
clustered regularly interspaced short palindromic repeats
what does Cas stand for
CRISPR-associated
explain how type II crispr system works
during initial viral infection: cas proteins recognize viral DNA @ the PAM sequence, chop up a protospacer (section of DNA), insert it into crispr array (an array of repeats of other chopped up DNA) as a “spacer” inbetween “crispr repeats”.
during subsequent viral infection: CRISPR loci (spacer) + part of crispr repeat are transcribed, transcripts processed to generate small CRISP RNAs (crRNA). crRNA hybridizes with tracrRNA (the crispr repeat is site of hybridization). This complex guides Cas9 endonuclease to invading DNA and recognizes it based on complementarity between spacer and target DNA, viral DNA is cleaved and degraded
what is the PAM
protospacer adjacent motif, NGG
in viral genomes this sequence is always present next to the protospacer, in the bacteria crispr repeat this sequence is not present
makes sure cas9 only cuts up viral DNA and not DNA
what does cas9 protein recognize?
DNA
what does cas13 protein recognize?
RNA
what is included in a vector to use for CRISPR?
2 oligos complimentary to desired target
cas9 protein sequence
gRNA
promoters
what to do if no nGG sequence near a gene you want to edit with crispr?
pick a different PAM, there are many proteins aside from cas9 that recognize other PAM sequences
what is the smallest cas protein?
cas 14, 400-700 amino acids in length
what to do if you want to remove a large sequence of DNA?
use two chimeric gRNA and Cas9 proteins to cut from either end
what is dCAS9? what can you use it for?
nuclease dead Cas9
use when you want to bind but not cut DNA, can attach an activation domain to activate a nearby gene, can attach an effector domain to make some chromatin or DNA mod, can add GFP to visualize gene
Can you think of potential practical application of the RNA-targeting Cas13/gRNA?
fuse with a fluorescent protein and see where RNA is localized
RNA knockdown
modify RNA to study it
antiviral defense (target viral RNA)
Why do you think the size of Cas proteins matters and what does Cas14 (~500aa) allow you to do experimentally that Cas9 or Cas12 (~1400aa) may not?
easier to transfect into smaller cells
viruses are small so cas12 wouldn’t fit
easier to build (shorter sequence to design)
bulky proteins can interfere with cell processes
less demanding of a cell to make smaller proteins
Why do you think in genome editing making two dsDNA breaks with the help of two Cas9/gRNA complexes may be preferred over making a single dsDNA break with one Cas9/gRNA complex?
to knockout multiple genes/a region of genes
make two cuts, if one guide RNA is less efficient than the other and goal is just to knockout and you don’t know which is most efficient co-express many and hope any cuts your gene
80% of cut are repaired without indels
