CFPS

Question 1

Give disadvs of genetic engineering

Answer 1

• Slow  Low efficiency rates
• Complex 
• Inconsistent, may not be reproducible

Question 2

Synthetic biology is what

Answer 2

designing/engineering new biological systems using standardised building
blocks; the design, build, test cycle; using artificially modified / synthesised DNA.

Question 3

What are the benefits of cell-free protein synthesis?

Answer 3

Benefits: Increased control, speed and scalability; reduced internal resource competition; more
quantitative;

Question 4

What are the opportunities brought

by cell-free systems?

Answer 4

can be taken outside the lab – different regulatory framework. - no GM legislation

Question 5

Give examples of DNA assembly protocols

Answer 5

o	BioBrick (Type I RE)
o	Golden Gate (scar-less) (Type IIs RE) – cut downstream of RS – scareless cannot be re-cut
o	Gibson Assembly  overlapping regions homologous can be synthesised into plasmids

Question 6

Explain the design build test cycle

Answer 6

within synthetic biology;
Design: In silico platforms aid biological design
Build: : lab atomisation facilitate complex assembly (quickly)
Test: measurement + extraction + analysis can be automated

Question 7

Give advs of synthetic biology in cells

Answer 7

Established methods
Self-replicate - cheap
Self-sustain
Compartmentalisation - membrane proteins + localised reactions and conditions

Question 8

Give disadvs on synthetic biology in cells

Answer 8

Established methods BUT poor reducibility, slow, restricted to evolve diversity

Self-replicate BUT strains must be purchased and maintained, growth does not scale linearly

Compartmentalisation BUT limited reaction conditions – homeostasis maintains constant settings, cofounding biological factors cannot be controlled + environment cannot be manipulated.
Toxic product biosynthesis is limited

Question 9

What are the 4 phases of CFPS?

What happens in each

Answer 9

1. Cell growth
2. Cell lysis
3. Extract clean up
4. CFPS reaction

Phase I Cell Growth
-Generates biomass from which cellular machinery is obtained

Phase II Cell Lysis
-Breaks cell walls to release cytoplasmic machinery/contents to be harvested

Phase III Extract Clarification
-Centrifuge, - Run-off reaction , - Dialysis, - Storage

Phase IV CFPS reactions - Transcription / translation operating outside of the cellular environment

• Supplemented
• Reaction incubated for protein synthesis to occur

Question 10

What supplements do CF reactions require

Answer 10

AA for protein biosynthesis, Mg, K and DTT to buffer reaction, PEG for molecular crowding, Energy solution for ATP regeneration, DNA template of target protein

Question 11

When was the first cell-free system developed? on what?

Answer 11

• Original cell-free systems developed in the 1950’s using rat liver cells

Question 12

List things to optimise CFPS

Answer 12

• Increase protein yield (1)
• Identifying factors impacting on extract activity
• Reducing CFE prep time
• Reducing costs
• Scalability

Question 13

What doesPURE system stand for

Answer 13

protein synthesis using recombinant elements

Question 14

What factors affect CFPS performance

Answer 14

• CFE extract prep affects performance
• Other factors impact of CFPS…
• Environmental conditions – higher fluorescent output seen at 37 than 25 degrees
• Reaction composition – increased concentration of reactants, increase fluorescent
• Genetic architecture – the DNA template being used– diff RBS’s contributed to diff GFP outputs

Question 15

What does genetic architecture mean

Answer 15

the DNA template being used in the CFS

Question 16

List applications for CFS

Answer 16

Outside of the lab – Gel stickers 
• Produce models 
• Diagnostic devices 
• Express toxic product 
• Agriculture 
• Pharmaceuticals

Question 17

What are the 4 diff types of systems often used

Answer 17

repressor
AND gate
toehold
ON system

Question 18

Explain repressor system

Answer 18

constitutive promoter (on all the time) producing protein A. Protein A binds to promoter controlling protein B and repressors it production  low amounts of protein B. When stimuli is added it releases protein A from the promoter if protein B  increased B expression.

Question 19

Explain AND gate

Answer 19

Both protein A and B are both required to switch on the promoter for protein C production.

Question 20

Explain toeholds

Answer 20

mRNA is produced, produces toehold region which releases the ribosome binding site from the ribosome. The ribosome cannot bind, therefore cannot get protein production. However in the presence of Trigger ligand is present it linearisers the loop, exposing the RBS site, the ribosome can then bind  protein production.

Question 21

Explain ON system

Answer 21

; protein A is produced however when protein A is by itself it cannot bind to protein B promoter, requires both trigger protein and protein A to activate protein B promoter. Protein B likely to produce a fluorescent promoter output.

Question 22

Which mechanisms are examples of transcriptional regulation and which are translational?

Answer 22

transcriptional - repressor and AND Gate

translational - toehold

Question 23

Give an example of a CF repressor system, by who?

Answer 23

Freemont group 2013
o LacR protein represses the production of GFP
o AHL binds to LacR and releases repression

note: optimised promoter (PLasR3) has the greatest output

Question 24

Give an example of an ON system? by who?

Answer 24

2019.

Benzoate required to switch system “ON”, sfGFP (superfolded GFP) when BenR protein expressed.

Question 25

how does a cocaine biosensor work?

Answer 25

Benzoate biosensor developed in 2019.
Cocaine -> Benzoate via CocE enzyme.

Increase in cocaine concentration – increase in fold change of GFP produced.

Question 26

Give an example of an environmental biosensor? by who?

Answer 26

e. g. banana, kiwi RNA
1. Extract RNA
2. Amplify RNA

• use a Toehold system; trigger ligand (amplified RNA) required to expose RBS for ribosome to bind.
  Can identify which ‘kiwi/banana’ based on the RFU outputs, specific outputs.

Question 27

Who developed paper-based CFS?

Answer 27

Pardee et al 2014.

• paper
• Freeze-dry for transport
• Reconstitute with water
• Toehold system
• Fluorescent protein output
• Colorimetric output; lacZ mediated colour change (in this case yellow -> purple) over time greater colour change

detects ebola mRNA

Question 28

who developed a CFPS hydrogel array?

Answer 28

Byun et al.2013 developed a CFPS array

-modifying agarose led to localisation of protein expression

Question 29

What are some applications of hydrogels?

Answer 29

Adhesives • Arrays and beads • Novel materials • Composite fibres

Question 30

Give an example of a potential use of a hydrogel?

Answer 30

e.g. self-disclosing systems, wound dressing detect bacteria present – release antibiotic. Field, crop detection etc.

Question 31

Give examples of cell lysis techniques

Answer 31

o Sonication cheap   evidence in literature that this method is more effective than other methods e.g. bead beating
o Bead beating  commonly used but other methods may be more effective
o High pressure homogeniser   expensive, not commonly used but effective
o Enzymatic (e.g. lysozyme)
o Freeze / thaw cycles  ice crystals rupture the cells

Question 32

Give an example of potential CFS

Answer 32

self-disclosing systems, wound dressing detect bacteria present – release antibiotic. Field, crop detection etc.