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Flashcards in Synthetic biology Deck (23):
1

What is synthetic biology

The design, construction, testing, and deployment of improved or novel biological systems using engineering design principles.

2

What are the three requirements for synthetic biology

Standardisation
Modularity
Modeling

3

How is synthetic biology standardised

Biobricks.
Specific enzyme recognition sequences removed so can be used to allow modularity (e.g. EcoRI/Xbal)

4

How is modularity achieved in synthetic biology

Biobricks can be changed around. Different promoters/RBS/ etc can be inputted. Changes output.

5

What is mathematical modelling and why is it necessary

Can tell if a synthetic gene is viable. Saves on wasted time/money.
Involves comparing the rate of transcription VS the rate of mRNA degradation. Rate of transcription needs to be more than rate of degradation for it to work.

6

Name some applications of synthetic biology

Medicine (Artemisinin, XNA)
Biotechnology (Biobutanols, Biodiesel)
Left field (Synthia, quadruplet code)

7

What is Artemisinin and why is it medically useful

Artemisinin is the best known treatment of malaria. It is naturally produced by plants, but is a low quality and yield.

8

How has Artemisinin been bioengineered to improve it

Bioengineered the plant by up/down-regulating specific enzymes, which increased the yield (OF ARTEMISINIC ACID)(to 1.6g/L).
More recently have up-regulated cytochrome b5 (enhancing cytochrome p450 activity) and introduced other enzymes into the genome. Yield has been increased to 25g/L.
Then is converted to artemisinin

9

How is artmisinic acid converted to artemisinin

Done chemically. Undergoes a photochemical conversion - UV causes conversion.

10

What is XNA and why is it medically useful

Xeno-Nucleic Acids. Synthetic DNA which contains 1,5-anhydrohexitol instead of ribose.
Could be used as Aptamers (short sequence that bind to proteins). Specific aptamers are able to deliver 'cargo' to specific cells (such as inhibitory RNAs). This is useful because XNA could potentially reduce side effects and avoid detection by immune cells

11

How were HNAs engineered

A polymerase was created that synthesises HNA from a DNA template.

12

What were the steps involved in engineering a polymerase than can synthesise HNAs

Polymerase - remove uracil stalling and 3'-5' exonuclease activity. General random mutation library of the polymerase, then test the mutants through Compartmentalised Self-Tagging to find one that can synthesise HNA.

13

What is the process of Compartmentalised Self Tagging as a means to isolate a particular polymerase

Each polymerase mutant is put in a separate E.coli cell.
Each cell is trapped in a droplet with an affinity-tagged nucleotide primer and HNA nucleotides.
Heat to break cells. polymerase plasmid hybridises with primer.
Affinity beads selectively capture primer + hybridised plasmids; wash out.
Plasmids remaining can synthesise HNA

14

Name some biofuels

Bio-
hydrogen
ethanol
methanol
butanol (**)
diesel (**)
methane

15

Why is biobutanol a good choice for an alternative fuel

Lower volatility
Lower hydrophilicity
More miscible with other hydrocarbons
Higher energy content per mass unit
(It doesn't mix well with water - easy to transport/use)

16

How has synthetic biology attempted to produce butanol biologically

By engineering e.coli to produce it naturally

17

How can e.coli be engineered to produce butanol

A natural e.coli amino acid biosynthesis pathway produces 2-ketovalerate as an intermediate - a precursor to butanol.
Synthetically added random 'ketoacid decarboxylases' hoping that one would form butanol. Tested levels of butanol produced (KivD - yeast - was best)

18

What is the traditional type of biodiesel

Vegetable oil - Triacylglycerides/triacylglycerols - broken down by an alcohol (meth/eth anol) which produces energy.

19

What does FAEE stand for

Fatty Acid Ethyl Ester

20

What is bad about the traditional method of making biodiesel

Uses lots of vegetable oil (starting product); and the alcohol (reactant) is usually a by-product from making petrochemicals (bad). Can also solidify in colder countries

21

How can synthetic biology help to improve the traditional method of biodiesel production

Can use e.coli to produce 'biodiesel'.
Naturally produces Pyruvate from glucose.
Synthetically added enzymes turn pyruvate into ethanol.
Synthetically added 'exogenous oleate' is naturally turned into oleic acyl CoA (through beta-oxidation)
Synthetically added enzyme is able to merge oleic acyl CoA with ethanol to form ES/DGATab which can be used as biodiesel!

22

What cool stuff can synthetic biology do (for fun)

Create synthetic organisms (Synthia)
Introduce a 4th codon into the traditional 3 codon reading frame.

23

What is Gibson Isothermic Assembly

A DNA assembly method which allows multiple DNA fragments to join in a single, isothermic reaction.
Requires exonuclease, DNA polymerase, DNA ligase
The DNA fragments must have about 20-40 bp overlap with adjacent fragments.
The exonuclease chews back from 5' end of fragments. These fragments can now anneal. DNA polymerase fills in any gaps. DNA ligase merges it all together.