Synthetic biology

Question 1

What is synthetic biology

Answer 1

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

Question 2

What are the three requirements for synthetic biology

Answer 2

Standardisation
Modularity
Modeling

Question 3

How is synthetic biology standardised

Answer 3

Biobricks.

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

Question 4

How is modularity achieved in synthetic biology

Answer 4

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

Question 5

What is mathematical modelling and why is it necessary

Answer 5

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.

Question 6

Name some applications of synthetic biology

Answer 6

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

Question 7

What is Artemisinin and why is it medically useful

Answer 7

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

Question 8

How has Artemisinin been bioengineered to improve it

Answer 8

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

Question 9

How is artmisinic acid converted to artemisinin

Answer 9

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

Question 10

What is XNA and why is it medically useful

Answer 10

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

Question 11

How were HNAs engineered

Answer 11

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

Question 12

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

Answer 12

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.

Question 13

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

Answer 13

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

Question 14

Name some biofuels

Answer 14

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

Question 15

Why is biobutanol a good choice for an alternative fuel

Answer 15

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)

Question 16

How has synthetic biology attempted to produce butanol biologically

Answer 16

By engineering e.coli to produce it naturally

Question 17

How can e.coli be engineered to produce butanol

Answer 17

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)

Question 18

What is the traditional type of biodiesel

Answer 18

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

Question 19

What does FAEE stand for

Answer 19

Fatty Acid Ethyl Ester

Question 20

What is bad about the traditional method of making biodiesel

Answer 20

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

Question 21

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

Answer 21

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!

Question 22

What cool stuff can synthetic biology do (for fun)

Answer 22

Create synthetic organisms (Synthia)

Introduce a 4th codon into the traditional 3 codon reading frame.

Question 23

What is Gibson Isothermic Assembly

Answer 23

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.