CFPS Flashcards
Give disadvs of genetic engineering
- Slow Low efficiency rates
- Complex
- Inconsistent, may not be reproducible
Synthetic biology is what
designing/engineering new biological systems using standardised building
blocks; the design, build, test cycle; using artificially modified / synthesised DNA.
What are the benefits of cell-free protein synthesis?
Benefits: Increased control, speed and scalability; reduced internal resource competition; more
quantitative;
What are the opportunities brought
by cell-free systems?
can be taken outside the lab – different regulatory framework. - no GM legislation
Give examples of DNA assembly protocols
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
Explain the design build test cycle
within synthetic biology;
Design: In silico platforms aid biological design
Build: : lab atomisation facilitate complex assembly (quickly)
Test: measurement + extraction + analysis can be automated
Give advs of synthetic biology in cells
Established methods
Self-replicate - cheap
Self-sustain
Compartmentalisation - membrane proteins + localised reactions and conditions
Give disadvs on synthetic biology in cells
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
What are the 4 phases of CFPS?
What happens in each
- Cell growth
- Cell lysis
- Extract clean up
- 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
What supplements do CF reactions require
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
When was the first cell-free system developed? on what?
- Original cell-free systems developed in the 1950’s using rat liver cells
List things to optimise CFPS
- Increase protein yield (1)
- Identifying factors impacting on extract activity
- Reducing CFE prep time
- Reducing costs
- Scalability
What doesPURE system stand for
protein synthesis using recombinant elements
What factors affect CFPS performance
- 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
What does genetic architecture mean
the DNA template being used in the CFS
List applications for CFS
Outside of the lab – Gel stickers • Produce models • Diagnostic devices • Express toxic product • Agriculture • Pharmaceuticals
What are the 4 diff types of systems often used
repressor
AND gate
toehold
ON system
Explain repressor system
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.
Explain AND gate
Both protein A and B are both required to switch on the promoter for protein C production.
Explain toeholds
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.
Explain ON system
; 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.
Which mechanisms are examples of transcriptional regulation and which are translational?
transcriptional - repressor and AND Gate
translational - toehold
Give an example of a CF repressor system, by who?
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
Give an example of an ON system? by who?
2019.
Benzoate required to switch system “ON”, sfGFP (superfolded GFP) when BenR protein expressed.
how does a cocaine biosensor work?
Benzoate biosensor developed in 2019.
Cocaine -> Benzoate via CocE enzyme.
Increase in cocaine concentration – increase in fold change of GFP produced.
Give an example of an environmental biosensor? by who?
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.
Who developed paper-based CFS?
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
who developed a CFPS hydrogel array?
Byun et al.2013 developed a CFPS array
-modifying agarose led to localisation of protein expression
What are some applications of hydrogels?
Adhesives • Arrays and beads • Novel materials • Composite fibres
Give an example of a potential use of a hydrogel?
e.g. self-disclosing systems, wound dressing detect bacteria present – release antibiotic. Field, crop detection etc.
Give examples of cell lysis techniques
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
Give an example of potential CFS
self-disclosing systems, wound dressing detect bacteria present – release antibiotic. Field, crop detection etc.
