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SynBio Flashcards

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1
Q

Where is a lot of compounds that we use today produced from?

A

Petroleum! Menthol, lavender and natural rubber is both produced via plants (harvesting) AND by petroleum production.

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2
Q

What is specialized metabolism?

A

The bio synthesis of specific compounds (specialized metabolites). Not a part of basic metabolism (such as Krebs cycle, FAO etc). Often used for protection of the plant itself.

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3
Q

What are some problems by using plants for the production of needed compounds?

A

The harvesting has limits. It uses space we could use for other crops. The extraction of the compounds from plants are environmentally bad.

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4
Q

How are terpenoids classified?

A

They all start as C5 compounds (5 carbons)
Monoterpenoids – C10 compounds
Sesquiterpenoids – C15 compounds
Diterpenoids – C20 compounds

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5
Q

What are monoterpenoids often used for?

A

Flavors/aroma in food and beverages. It is not sustainable the current production. So we have to use synthetic biology and produce in for example in yeast.

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6
Q

Isoprenoids are what?

A

Terpenoid precursors and are present in most cells like yeast from the basic metabolism. (GPP,FPP)

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7
Q

Yeast uses what compounds for growth?

A

Sterols! They need sterols from growth, so remove FPP and it dies. - Competition!

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8
Q

What is orthogonal?

A

Orthogonal means independent (pathways can be orthogonal)

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9
Q

What is dynamic regulation/control?

A

Control pathway and how much it should produce according to the cell or us. Only works if it is orthogonal. You introduce a metabolic valve to induce this control.

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10
Q

What is PTS1 or PTS2?

A

Peroxosmal Transport Signal (or something, check abbreviation) 1 or 2, makes sure the protein in send to the peroxisome.

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11
Q

Insulating production in peroxisome does what`?

A

Increases production of your desired product, as the substrate will enter the peroxisome, but will not have competition by other enzymes, so your production enzyme will have it for itself without competition.

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12
Q

What are the key elements in Synthetic Biology Design?

A

Chassis, Modules, Devices and Parts

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13
Q

What are the key concepts in SynBio?

A

Abstraction, Modularity and Standardization

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14
Q

What are the key enabling technologies for Synbio?

A

DNA synthesis
Next generation sequencing
High throughput cloning techniques
Genome editing technology (TALENS, CRISPR)
Modeling of Biological systems
Repositories of Standardized parts (BioBricks)

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15
Q

What are the key assisting technologies for Synbio?

A

Modelling and Computer Aided Design of Biological Systems
Development of Directed Evolution methodologies
Developing of Metabolites Chemical Analysis
Upscaling/Fermentation

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16
Q

Why are biorefineries/bioproduction/synthetic biology production good?

A
  • Sustainable, based on metabolic systems via metabolic engineering and not petrol-based synthesis.
  • Can produce everything from insulin to biofuels.
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17
Q

Metabolic pathways of importance are?

A

Glycolysis, FAO, Acetyl-CoA can be turned to GPP into monoterpenes.

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18
Q

Why are tobacco plants good?

A

Transient expression is fast, easily transfected

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19
Q

What are the pros of using plant chassis for SynBio?

A
  1. Autotrophic, use light and CO 2
  2. Free of ethical issues vs animal cells
  3. Plants can produce energy, food, medicines
  4. Agriculture sustainability
  5. Plant cells are generally not hosts of human pathogens
  6. production of pharmaceuticals and high value products
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20
Q

What are the cons of using plant chassis for SynBio?

A
  1. GMOs legislation
  2. Complex organisms
  3. Not enough tools (today, but we are getting better)
  4. Slow life cycle
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21
Q

What are the main plant chassis used today?

A 
Nicotiana Benthamiana/Tabacum
Moss
Algae
Arabidopsis thaliana
Liverworts
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22
Q

What are the advantages of moss?

A
  • Small
  • Absorb water and nutrients through leaves
  • Non-vacular/water bearing vessels
  • Can be transformed
  • Scalable
  • Sequenced genome
  • Homologous recombination
  • Synthetic biology and DNA assembly techniques
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23
Q

What are advantages to algae?

A

Autotroph
Can be transformed, nuclear/chloroplast/mitochondrial transformation
Scalable
Unicellular, so lower complexity
Homologous recombination
Already developed CAD softwares
Do not compete with food crops for land/resources

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24
Q

Agrobacterium can?

A

Can invade the plant cell and genome and deposit DNA material. Is normally carcinogenic for plants, but in the lab we remove the harmful genes and inserted the gene of interest in a plasmid that will be transferred.

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25
Q

Biolistic Method is what?

A

Shoot plants with small particles covered with the desired plasmid. Not very efficient.

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26
Q

What are hairy roots?

A

Cellular hair. Roots generated by the infection of an agrobacterium that forces the plant to produce a lot of this hairy tissue – “hairy roots”. Can be used with specific genes. They loose stability over generations.

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27
Q

Plant cell suspensions are?

A

Plant cells with the plant wall removed with enzymes.

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28
Q

What is specialized metabolism? What is general metabolism?

A

General: Photosynthesis respiration for growth and reproduction
Specialized: Synthesis of metabolites used for interaction and adaption in the environment

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29
Q

What important discoveries enables synthetic biology?

A

Sequencing, synthesis of DNA, automation

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30
Q

We used to produce synthetic DNA in 96 well plates. What do we produce in now?

A

Microarray based chips with thousands of microscopic wells.

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31
Q

What is the double-sided issue with the breakthrough in DNA reading?

A

We are very good at reading DNA, but not very good at writing DNA yet (ie. It’s expensive to synthesize DNA still).

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32
Q

Mention natural products that can be produced by SynBio

A

Terpenoids, Alkaloids, Cannabinoids, Vanilin

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33
Q

Why is Cytochrome p450 essential?

A

It is essential for chemical diversity driving Oxidation and:

  • Hydroxylations
  • Dehydrogenases
  • Glucosyltransferases
  • Methyltransferases
  • Acyltransferases
34
Q

What is a sequencing library?

A

A preparation of the DNA before sequencing. Like: 1) Fragment DNA. 2) Add platform-specific adapters (so the machine knows where the sequence starts and ends)

35
Q

What is directed evolution?

A

Mimicking of natural selection but on selection on variants based on artificial selection conditions. (Like extremely high conc. Of antibiotics).

36
Q

Why is DE smart and how can you do it?

A

Induced mutagenesis
Random or targeted mutations
Selection based on gene level
• Short generation time

37
Q

What can be evolved in DE?

A
  • Protein engineering
  • Metabolic engineering
  • Production organism engineering
  • Gene properties
  • Enzyme properties
  • Protein physical properties
38
Q

What are methods for inducing DE mutagenesis?

A

Error-prone PCR
Site saturation libraries (libraries with all AA codons on a spot etc.)
Transposon mediated mutagenesis (insertional mutagenesis)
DNA modifying enzymes (deaminases, glycosylaser)
CRISPR/Cas9 mediated methods (both editing + modifications)

39
Q

How do you control DE?

A

“You get what you screen and select for” – what for “cheaters”. Antibiotic resistance maybe? – NO! Could be blocking it from entering the cell.

40
Q

Membrane lipids have what physical ability that makes cell membranes so stable?

A

They are amphiphilic (they both like and dislike water/hydro-phobic and -phillic), so the hydrophobic parts wants to stay together and the phallic parts wants to stay together (because of entropi).

41
Q

What lipids are in the cell membrane?

A

Glycerolipids, Sphingolipids and steroids. They are all present in plants and animal cells, but Glycerolipids is the only lipid in bacteria.

42
Q

What can glycerophospholipid have?

A

They can have negatively charged or non-charged phospholipid head groups! Artificially, you can make positively charged heads, but they are not present in nature.

43
Q

What is the structure of glycerophospholipids?

A

A head group and two fatty acid chains. You always have two fatty acids, and one of them is often unsaturated (that is, they have one or more double bonds, which are less mobile)

44
Q

What are bolalipids?

A

Lipids that span the entire membrane (a head group-fatty acid-head group). Because of this head-FA-head structure, it is extremely stable, so good for high temperatures, but maybe not as permeable.

45
Q

What determines which structure lipids in water will form?

A

The shape of the lipid. The head size vs. fatty acid chain size will contribute to whether it becomes a bilayer, micelle, inverted micelle or liposome etc.

46
Q

What is the difference between membranes in the body?

A

They all have different compositions of lipids so that they get different abilities.

47
Q

Thickness, curvature, intracellular transport and membrane fluidity and mobility is defined by?

A

The lipid type. Thickness = longer fatty acid chains (C16 vs. C22).
Curvature = different shaped lipids.
Intracellular transport = make inside lipid differently charged than outside lipids, inside could be negatively charged and outside neutral.
Fluidity and mobility = higher number saturated vs. unsaturated steroids and steroids in general.

48
Q

What happens when you mix a lot of different lipids?

A

They organize into microdomains based on the type of lipid.

49
Q

Extruders can create what size of liposomes?

A

Liposomes the size of the pores in the extruder that pushes through the lipids.

50
Q

What is the nanodisk system?

A

Helper proteins with a hydrophobic and -phillic sides. Inside this helper protein is lipids and the protein of interest.

51
Q

Why is nanodisk smart?

A

Because it gets water soluble! You can do everything you could do on a cytosolic protein.

52
Q

Describe how you typically would isolate membrane proteins.

A

1) Solubilization/purification via detergents
2) Lipid addition
3) Remove detergent

53
Q

What is the thermodynamically most stable conformation of a protein?

A

The energy minima (on the bottom of a conformation/energy curve) – the most stable configuration.

54
Q

Are proteins static? And what is this called?

A

No! They fluctuate in different conformations and getting past different energy barriers into the next stable conformation. The lower the energy barrier, the faster the transition, and the higher the barrier, the higher the transition time. This is called “The Energy Landscape”, can also be thought of as a landscape or a weirdly shaped funnel (with dibs in it).

55
Q

What causes fluorescence?

A

An atom is excited and enters a higher vibrational energy state, this can be release as photons and fluorescence. OR the vibrations can dampen over a long time.

56
Q

Is protein activity static?

A

Proteins fluctuate in activity on the microscopic scale.

57
Q

If you get 5 active conformations of a protein, how do you tell the cycle of them?

A

You can determine the different stages of the protein by using FRET. You can measure how close different chomophors (fluorescent proteins) on the molecule are. The donor chomophor will donate som of its energy to the acceptor chomophor which will absorb some of the energy and emit another wavelength. You can now record this fluorescence like a movie and see the change in proximity of the different areas of the molecule. This change in fluorescence is handled by Ai software and you can see that conformation 1 is active for X nanoseconds, conformation 2 is active for X nanoseconds etc. You can then see the specific order of them, that it goes from 1 to 3, 3 to 2 etc.

58
Q

How can you engineer proteins to stop entering a specific conformation (that for example will not cut DNA?)

A

You can introduce mutations that will make it harder to enter the specific conformation by increasing the energy barrier.

59
Q

Working efficiency and working hours in these single molecule FRET experiments showed by?

A

Movement = working and moving speed shows working efficiency. If it’s not moving, it’s not working, if it’s moving fast, it’s working efficiently.

60
Q

Describe GPCR signalling

A

Agonist binding –> G protein coupling to receptor and nucleotide exchange (GDP exchanged with GTP) –> Subunits decouples from Receptor –> Activated G protein subunit activates effector proteins –> Adenylyl Cyclase makes cAMP from ATP + Ca2+ influx is activated through binding to a L-type Ca2+ channel.

61
Q

Types of GPCR ligands

A

Neutral antagonist – binds to receptor but has no effect on the basal activity.

Partial agonist – stimulate the effector proteins to less than maximum. Tend to have higher affinity, but will not activate to the full poteintial.

Full agonist – stimulate the effector proteins to the maximum.

Inverse agonist – Binds and blocks receptor but stabilizes the inactive state as well! So, the activity falls.

62
Q

GPCR properties

A
  • 7 transmembrane domains of alpha helix
  • Extracellular N-terminus
  • IC – C terminus
  • Ligand binding domain
  • Domain important for G protein coupling.
  • Can signal through Arrestin in the endosome (When activated, GPCR’s can be phosphorylated, arrestin can bind and GPCR can be internalized clathrin-mediated, in the endosome arrestin can signal through the ERK/MAP pathway.)
63
Q

GPCR oligomerization:

A
  • Can be monomers, heterodimers, homodimers or even oligos!
64
Q

Nanobodies are?

A
  • Single domain antibodies from Llamas, camels and dromedroids
    o They do have normal Ab as well
  • Are Ab that lack the light and heavy chains and only have the hypervariable region.
  • Can bind hidden antigens that are not accessible by whole antibodies (such as active sites of enzymes).
    o Especially because the nanobody has a long antigen binding loop that is longer than conventional Abs.
  • Can be used to stabilize proteins in a certain active state.
  • Can be expressed in bacteria and produced MUCH more easily than Abs. Abs needs to immunize mammals and then harvest and you can’t produce more.
  • Good tissue penetration, so can be used in medication (but will be filtered out in your kdineys, so it’s needs to be a fusion protein above 50 kilodaltons)
65
Q

Advantages of biosensors are?

A
  • Specificity
  • Sensitivity
  • Price
  • Speed
  • High throughput
  • Integration into production strains
66
Q

What is the anatomy of a biosensor?

A

1) Analyte
2) Sensing entity
3) Transducer
4) Processor
5) Displar/reporter (Fluroescence, color, luminescence (luciferase)

67
Q

What can biosensors sense?

A
  • Ligands to Membrane receptors
  • Nucleic acids
  • Transcription factors
  • Enzymes/ligands
  • CRISPR
  • Antibodyes

Chemicals, anything really.

68
Q

What is dual use?

A

Technology that can used for development of biological weapons. (Like developing pox virus for research, that could also infect humans).

69
Q

Recombinases can do what?

A

Invert a dsDNA and turn on a gene so you can get a on/off state.

70
Q

What is different from digital and analog computing?

A

Digital computing (also in cells) is divided into 0’s and 1’s whereas analog signals are based on a gradient. Could be conc. Gradients.

Memory can also be added to organic circuits.

71
Q

What is photosynthesis at its most basal level?

A

Excitation of pigments by light.

72
Q

Oxygeninc photosynthesis is?

A

Light into electrons and ATP and finally into organic molecules

73
Q

Why is photosynthesis and photosynthetic organisms relevant in synthetic biology?

A
  1. Access to carbon skeletons (terpenes!)
  2. Energy and electrons in a sustainable way; solar radiation, water and carbon dioxide
    - -> We can use it to drive biosynthetic reactions
74
Q

How can we drive biosynthetic reactions via photosynthesis?

A

Electron from PSI (light sensitive part of photosynthesis) can be transferred to P450 for further reactions.

75
Q

P450 has reduced productivity because of electron “stolen” by other plant components. What can be done for this?

A

Merge P450 to ferredoxin, this yields more e- to p450

76
Q

What can also be produced via photosynthesis-driven biosynthesis?

A

Aromatic amino acids – High demand for food and pharma, bioactive compound precursors, food and feed supplements.

77
Q

Ideal traits for a cell factory industrial production are?

A 
Grow in cheap feedstocks
Fast grow and high yields productivity
Shape suited for bioreactor cultivation
Shielded ” against shear stress
Amenable to genetic modification
High secretion capacity
Preferably eukaryotic system (protein production
Easy to clean
78
Q

What are the three fermentation modes?

A

Batch (Isolated)
Continuos (feeding + output)
Fed-bacth (Feed and no output before “harvest”. Avoids inhibiting effect of high initial subsrate, can reach high titers)

79
Q

DE is up and down because?

A

It’s a landscap. You might have to go back form your peak, to reach the actual highest peak.

80
Q

Types of biosensors?

A

Biophysical
- Immobilized enzymes
- Antibodies
Cell free systems (proteins)

Whole cell

  • Engineered microbes
  • Mammalian Cells
  • Plant/Algae/Yeast

Tissue

  • Engineered fish
  • Tissue cultures
  • “Organ-on-a-chip”

Synthetic Biology (1 decks)

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