Synthetic Biology Flashcards
(25 cards)
Normalising data equation
I (normalised) = I - I(min) / I(max)- I (min)
ASO conjugates
Anti-sense oligos
ssDNA that bind to mRNA and block translation/cause degradation/change splicing
example for tracking AND targeting > ASO links fluor dye, and targeting peptide
Dangers of conjugation
1) overconjugation - risk destroying acitivity of target
2) labelling distribution - have to find sweet spot in amount of label to add - labelling and activity not always correlated
3) purification - how clean is the final product - is there residual unbound label OR leftover unbound molecule of interest?
chemical transfection
Chemicals include: (1) cationic polymers that have a positive
charge which binds to DNA and neutralises its negative charge, allowing it to enter the
cell and (2) lipids that are externally hydrophilic and internally hydrophobic encase the nucleic acid, forming liposomes which can easily transverse the cell membrane.»_space; overcomes many other techniques shortfalls!
Key size nanoparticles
<100 (kidney), tend to coalesce - fuse together
>100 (liver/spleen), unstable - want to eat up smaller particles
100nm is the “magic size”
test for size using DLS
DLS
Interaction of ___ with the ___ ___ of a small particle
Dynamic Light Scattering
light, electric field
Scattering Theories
Rayleigh
- applies when the particle diameter is less than 1/10th of the wavelength
- light is scattered roughly equally in all directions
- intensity of the scattered light is proportional to the size of the particle ^ 6
Mei
- for particle sizes larger
- directional dependence of the scattering is no longer uniform and changes depending on particle size
- exact complicated mathematical soluition
Brownian motion
> random movement of particles due to collisions with solvent particles
faster when:
> smaller particles
> less thick soln
> higher temperature
Correlation (extracting time dependence of the signal)
larger/smaller curves look like?
Larger particles: shifts the function to the RIGHT (picture looks the same for longer as particles move more slowly)
Smaller particles: shifts the function to the LEFT (vice versa)
Y-intercept
Gradient
Baseline
- if intercept is low, particles are too small to measure
- should be steep, if shallow or wobbly then many different sizes
- should be flat, if noisy there may be large particles (dust) present
Intensity, volume, number relationships
I = N x D^6
V = N x D^3
Limitation of DLS
> large particles mask smaller ones
dust is large (1mm) - results in greater scattering which masks nano particles
Properties of Polymers
> polymer length/weight per chain (kDa) affects physical properties viscosity, stiffness, malleability
> dispersity (variation in chain lengths) affects viscosity, strength, resistance
> architecture (branching) different side chains give different properties
Pros and Cons of synthetic polymer SYNTHESIS
PRO:
- biodegradable polymers
- build complex sequences
- DNA template
CON:
- backbone limited by AA properties
- can only build short chains
- still need to power/feed bioreactors
Pros and Cons of synthetic polymer DEGRADATION
PRO:
- break down non-biodegradable polymers
- nature can adapt to pollution
CON:
- bacterial growth and polymer usage not on same scale
- need to speed and scale up process
- unknown by-products
Literature e.g.s of polymer synthesis and degradation
SYNTHESIS
Sense codon reassignment
- recode codons, provide synthetic tRNAs as replacements, results in viral resistance
DEGRADATION
bacteria degrades PET plastic
- screened from nature, maybe some lab evolution, test breakdown products
Size Exclusion Chromatography
Separation based on sieving
For molecular weight determination of polymers
Large molecules elute first, smaller get stuck in pores and hang around
Cross linked gel (stationary) and solvent (mobile) phases
SEC distribution weighted averages
Mn (equal number of molecules each side)
Mw (equal masses of molecules each side)
Mz (biased toward large molecules)
SEC dispersity
Dispersity = Mw/Mn
tells how uniform the sizes are, narrow if between 1 and 1.2
Monodisperse protein still have broad SEC peak because:
> non-zero sample volume
> diffusion
> rel between size and pore accessibility
High Performance Liquid Chromatography
Sample onto column
most common: reverse phase
NON-polar stationary phase column
POLAR mobile phase solvent
thus, more polar elute first, first peaks, and height of peaks indicate amount of component
How to detect:
1) drugs/synthetic molecules
2) gene expression
3) cells that have received delivery
1) tagging with a fluorophore
2) fusion of reporter genes/ AB genes for selection
3) flow cytometry
Migration in gel elec influenced by:
Uses of gel elec:
size, shape, charge, chemical composition
determine number, amount, mobility of components, or separate them
determine electrical double layers
mol weight
structural changes in shape
Compensation to correct spillover
Typically done before sample aquisition
Uses unstained and sinlge colour stained sample controls as references in the estim of the degree of single interference
compensate with samle as bright/er than your sample
treat controls in the same manner as tests
Applications of Flow Cyt
Cell cycle (dye that binds DNA)
Cell viability (dyes in cell have different signal when dead)
Protein expression (fluorophore tagged antibody)
Gene delivery and editing (confirm delivered, change in signal)
