Five Hard Truths

Question 1

List the 5 hard truths

Answer 1

1. Many parts are undefined
2. The circuitry is unpredictable
3. The complexity is unweildy
4. Many parts are incompatible
5. Variability crashes the system

Question 2

What could be the solution to the fact that many parts are undefined?

Answer 2

Just need to do more work to characterize and standardise the parts.

Question 3

What are some solutions to unpredictable circuitry?

Answer 3

1. In silico (computational modelling) - work out how strong the ribosome binding site is - characterising strong/weak promoters
2. Directed evolution (tuning existing factors or creation of new functions by changing the sequence of the part) - random mutagenisis (error prone PCR, UV, chemicals), - site directed mutagenisis, DNA shuffling

Question 4

What are some solutions to unweildy complexity?

Answer 4

1. If more standards had existed then the work would have been carried out faster
2. Introducing more and better/different assembly methods/standardisation according to physcial composition

Question 5

What are some solutions to parts being incompatible with their host and reducing interactions?

Answer 5

1. Selecting the right chassis (E. coli vs yeast or anything else)
2. Engineering the chassis - tuning expression of toxic metabolites, silencing pathways
3. Design of orthogonal systems
4. Physical isolation of processes in cellular compartments (scaffolding)
5. Bottom up design strategy

Question 6

How could you map metabolic flux to assess metabolic burden?

Answer 6

By measuring the protein activity within a chassis using mass spectrometry to map the pathways. This is done by comparing the edited chassis to the original chassis and seeing where the differences lie to assess the burden.

Question 7

What are some solutions to variability crashing the system?

Answer 7

Engineering the chassis for stability

1. Deletion of mobile (viral) elements
2. Increasing the efficiency of polymerases so you don’t get errors and therefore mutations
3. Reducing recombination hotspots

Question 8

What causes variability?

Answer 8

1. Biological noise (heterologous population - not all the cells are in the same stage of cell division or growth, which results in inconsistent behaviour and mutation accumulation)
2. Biological instability - viral infection of bacteria/yeast where viral molecules integrate themselves into the actual genome of the cell and introduces mutations