Cell biology Flashcards
How does cellular diversity arise?
What does it produce?
What happens in the nucleolus?
What do peroxisomes do?
What 2 parts of the phospholipid membrane can change?
How does heat change the consistency of the membrane?
Asymmetric cell division from inter/intracellular signals
Cell lineage into differentiated cell types
rRNA processing & ribosomal assembly (for translation)
convert hydrogen peroxide into water
head groups in phospholipids
cholesterol- widens the membrane
gel like to fluid like
What are the pros and cons of light microscopes?
What stain is used and to what does it bind to?
Why is phase contrast light microscopy better?
Why is de-convoluted phase contrast light microscopy EVEN better?
How is fluorescence spectroscopy recorded?
Pro: easy to use, quick, cheap
Con: only for large cells/tissues, have to slice specimen thinly (dead), stain
Haemotoxylin (basic amino acids) & Eosin (acidic molecules/amino acids & DNA)
Has additional phase plate so takes advantage of different refractive indexes of contents of cell- means can look at living cell & get clearer picture
Phase plate & refinement so light is split into perpendicular indices- allows for 3D image & can use thicker specimens & assemble images to make whole organism
Lenses & mirrors focus the fluorescence & the beam-splitting mirror makes sure only emission energy is recorded at eyepiece
(more light microscopes)
How does confocal microscopy produce a sharper image of a thick specimen?
How does deconvolution microscopy work?
Uses fluorescence to narrow the focal plane & build up very thin images into a sharp 3D image
Stain cell with different dyes & use computer software to separate the light frequencies for clearer image
How are electron microscopes physically different to light ones?
What’s the difference between SEM & TEM?
What is the problem with CyroEM? How do you counter this?
How is CryoEM better than SEM/TEM?
How do you produce a GFP fusion protein?
What is FACS used for?
How does this work?
Beam of electrons instead of light & magnetic coils instead of mirrors- lenses in both
SEM = lower resolution (10nm), higher depth & field of view, easier, quicker, build up 3D image from electrons reflecting
TEM = high res (1nm), lower depth & field of view, more skilful & slow, allows electrons to go through specimen
Electron irradiation means break chemical bonds & form free radicals which damage sample.
Use heavy metals (absorb/deflect energy of electrons) and put in low temp (liquid nitrogen & helium) which improves resolution
higher resolution so at atomic level
make recombinant protein with DNA for protein of interest & insert DNA to code for GFP
sorting a cell type from a mixture using fluorescent fusion proteins
- mark desired cells with fluorescent protein (in fusion protein) & grow the cells in culture
- inject cell suspension & narrow funnel passes cells out 1 by 1
- fluorescence given out & recorded based on size & shape of the cell
- the spectrum of different fluorescences is used to apply negative charge relative to their shape & size- deflecting cells at different degrees relative to their fluorescent signal (hence shape & size)
What are 2 features of restriction enzymes in genetic engineering?
What were they originally used for?
What 3 different sticky ends can be generated?
What is an isoschizomer?
What enzyme sticks sticky ends back together?
What concept is key with sticky ends?
Cut short sequences of double stranded DNA = sticky ends
Palindromes- read same in forward & backward direction
bacterial defence mechanism against invading viruses
5’ overhang (5’-A x AGCTT-3’)
3’ overhang (5’-CTGCA x G-3’)
blunt (5’CCC x GGG-3’)
restriction enzyme that cuts at the same sequence but at the other prime overhang (e.g KpnI cuts 3’ overhang & Aspn718 cuts at 5’ overhang of same sequence)
T4 DNA ligase (re-establishes phosphodiester bonds with 2 ATP)
compatibility- don’t always need to cut with same restriction enzymes
What are 4 features of plasmids that make them useful for recombinant DNA?
What does the polylinker region contain? as a result what is it used for?
When inserting recombinant plasmids into E.coli, what can you do to allow transformation?
what is it called when a bacteria can naturally take up a plasmid from its environment?
how would you go on to harvest the recombinant DNA after transformation?
how can a phage be used as a vector (instead of a plasmid) to produce recombinant DNA?
extrachromosomal (circular DNA)
contain antibiotic resistant genes
replicate independently of bacterial DNA
replicate autonomously
many restriction enzymes- inserting DNA
mix with presence of CaCl2 (alters permeability of cell membrane)
competent
- clone DNA by growing a colony that survived on antibacterial plate
- harvest the cells, spin it down into pellet & lyse
make recombinant phage DNA & insert it into its head- phage will then inject DNA into the host bacterium through its tail
What is an expression vector?
In what cells can these be used?
what is transfection?
what type of DNA is inserted into the expression vector/plasmid?
what are the 2 methods of transfection and what are their pros/cons?
how can you induce the plasmids to enter the cells?
Vector (often a plasmid) containing promotor region to transcribe/translate sequence protein product from inserted cDNA
mammalian cells
introduction of foreign DNA into host cells
cDNA
- transient: genetic material remains extrachromosomal (on plasmid) and does not incorporate into host- therefore product is made but often plasmid is degraded
- stable (transformation): DNA incorporates into host genome so it’s stably retained & expressed- often requires antibiotic resistant gene
lipid treatment (so membranes fuse) or electroporation
