Reka Toth Flashcards
biolistics
important in a non-model species without approved transformation protocol
mechanical stimuli
not reproducible
how can you probe a pathway?
- receptors
- sensors
- kinases
- interacting proteins/co-factors
how do you compare between species
- transcriptomics and DEGs
- heterologous complementation assays
- think necessary and sufficient, and then phylogeny
- draw attention to the different between homology and essentiality
- compare between 3 to find an intermediate stage
double mutants show
genetic epistasis
if they have tested expression of genes
have they tested accumulation of product?
have they mentioned any signalling pathways which
they did not test?
pleiotropic defects
- if you’re using a reporter, have they tested for this? important to note; can cite this experiment
- don’t know how much of the observed response is actually due to the transgene
- hard to avoid if you’re working with something that affects many pathways
how many genes have they tested?
and are they in the same gene family?
be careful of confusing
- the expression of a gene regulator and the
- expression of the genes that they regulate
if they are investigating a particular stress response
does the responder affect many different stress pathways?
if they mention genes
have they functionally annotated them?
RNA methods
- RNA Seq
- qPCR
Protein detection and protein-protein interaction
- western blotting
localisation
- fluorescence
- immunohistochemistry
- GUS assay
DNA binding and epigenetic
- chromatin immunoprecipitation (ChiP-Seq)
gene mapping
- GWAS
- QTL
What is protoplast generation?
- isolating cells without their cell walls and introducing new genetic material
- gene editing, plant transformation, and cell culture studies
- Somatic cell fusion
transient expression
- Agroinfiltration and protoplast transformation
GC-MS
for chemical detection
flow cytometry
Cells or particles can be labeled with fluorescent dyes or antibodies that emit light of a specific wavelength when exposed to a laser
How does co-IP show protein detection or protein/protein interactions?
- pull down a specific protein and identify interacting partners
- An antibody that specifically recognizes the target protein
- eluted proteins can be analyzed by methods like Western blotting, mass spectrometry
- Mapping Signaling Pathways
- Studying Post-translational Modifications (PTMs)
What is GC-MS?
- used to identify and quantify compounds in a sample
- pesticides, herbicides, volatile organic compounds
How does yeast 2 hybrid (Y2H) show protein detection or protein/protein interactions?
- reconstitution of a transcription factor in yeast that is split into two parts: the DNA-binding domain (BD) and the activation domain (AD)
- If these two proteins interact with each other, the transcription factor is reconstituted, leading to the expression of a reporter gene
- LacZ
- Negative controls
- Mapping Protein-Protein Interaction Networks
- Identifying Novel Protein Interactions
How does immunohistochemistry show localisation?
ABS
How does chromatin immunoprecipitation (ChiP-Seq) show DNA binding and epigenetic effects?
- Cells are treated with formaldehyde or other crosslinking agents, which covalently bond proteins to the DNA. This “locks” protein-DNA interactions in place, ensuring that transient or weak interactions are captured
- fragmented chromatin is incubated with an antibody specific to the protein or epigenetic modification of interest
How does GWAS work?
- identifying a phenotype you want to study
- population: large and diverse to represent a variety of genetic backgrounds
- collect DNA and genotype for SNPs
- compare SNPs between cases and controls
How does QTL work?
- two genetically distinct parental lines
- measure the phenotypic variation for the quantitative trait in the offspring population
- genotype using molecular markers; tracks inheritance
- linkage mapping + ANOVA
Somatic cell fusion
Protoplasts from two different plant species can be fused to create hybrid cells with genetic material from both parents (useful in plant breeding).
Protoplast transformation
- electroporation uses an electrical pulse to create temporary pores in the protoplast membrane, allowing the foreign DNA to enter the cell
- Gene function analysis
- GE
How does mass spectrometry work?
- Protein Extraction
- Peptide Sequencing: tandem mass spectrometry (MS/MS)
- protein database
