cell-free biology

Question 1

define synthetic biology anddiscuss why it is used

Answer 1

-designing and constructing biological entities such as enzymes and and genetic systems outside cells

it is used because other biological methods are flawed; with selective breeding there is no control over which genetic material is transferred and with genetic engineering there are problems with speed, confounding factors and inconsistency

use of the design, build, test cycle

Question 2

how is synthetic dna assembled

Answer 2

1) biobrick assembly: standardrestriction enzymes used on gene and vector allowing assembly via sticky ends
2) golden gate assembly: secondary restriction sites are used which cut downstream of the recognition site and mean the same cut site cant be cut again by the same enzyme
3) gibson assembly: dna with overlapping sequences are ligated together using PCR

standardiation
automation

Question 3

synthetic biology can be carried out in cells. describe why cells are used (and the limitation of cell based systems)

state the advantages of cell-free systems

state the disadvantages

Answer 3

-established protocols exist for genetic transformation etc (poor reproducability and can be slow)
-organisms self replicate and self-maintain so are cheap and scalable (strains must be purchased and maintained)
-cell compartmentalisation allows for localised reactions (its difficult to manipulate interanl conditions)
(many confounding factors exist)

• control over systems (which genetic templates are present, which reaction conditions are occuring)
• linear scalability
• speed of systems; cells dont need to grow
• conversion efficiency is higher because there are reduced competition and confounding factors
• system can be taken out of the lab due to less stringesnt regulation
• practises arent well established; unforseen problems occur
• higher costs can occur due to preparation of extracts and use of specialised equitment

Question 4

describe how cell-free protein synthesis is done

Answer 4

1)grow a large number of cells to obtain cellular machinery
2)lysis of cells to release cytoplasmic contents using sonication, enzymes, high pressure or freeze/thaw cycle
(done during exponential growth to get highest amount of ribosomes or stressed cells which have a bugger range of transcripton factors)
3)clean up of extract from debris using centrifugation. DNases to break up remaining nucleic acids
4)extract is combined with building blocks of proteins (AAs, buffers, ATP, DNA template)
5)incubation and checkups for product

Question 5

how can cell-free systems be optimised

Answer 5

increase protein yield by changing when cells are harvested/clarification steps

• reduce extract preparation time;reduce cell-wash and dialysis steps
• reduce cost by avoiding use of specialist equipment
• improve scalability

pre-mixed extracts exist but are very expensive and there less options for optimising the buffer, some are very expensive

it is impractical to explore all the combinations of different methods of optimisation

trade offs exist; cost, yield, speed, reproducibility

Question 6

describe the different gene networks that exist in cell-free biology

Answer 6

• repressor; protein A binds to protein B promotor but is released to a stimuli
• AND gate; both protein A and B are needed to activate transcription of protein C
• toehold; mRNA forms a toehold region when being expressed which stops translation (ribosome RBS site moves) a ligand is needed to linearise the toehold region
• ON system; protein A can only bind to protein Bs promotor when triggered

Question 7

describe using cell free biology in materials

Answer 7

• reaction occur on a solid matrix
• diagnostics; when different strains of ebola are attached to paper, different florescent responses occur due to differing RNA