Lecture 2. How to Design and Build Novel Genetic and Biochemical Systems Flashcards
What are the problems of realising synthetic design?
Variable properties of the circuitry components
Circuit-host interactions (degree of orthogonality, make sure synthetic system doesn’t interfere with cellular function)
Gene expression noise / cell-to-cell heterogeneity
What are the two complementary approaches to enable predictable synthetic circuitry construction?
Characterise quantitatively the properties of circuit components and use this information to improve their performance
Incorporate the outcomes of the above experimental strategies into more advanced computational algorithms in order to facilitate design and construction
What must also be accounted for when quantitatively characterising the properties of circuit components?
Characterising noise / cell-to-cell heterogeneity and develop ways to control (suppress) negative noise effects
What must be ensured to have functional coupling between two promoters, where one promoter generates a transcription factor (TF) that activates or represses the activity of the second promoter?
Need to ensure that the range of concentration of the TF generated by promoter 1 (the output) matches the input sensitivity range for promoter 2
What does the Hill equation show?
Fraction of binding sites occupied by ligand (TF) as function of unbound ligand concentration
What is the Hill equation formula?
x^n / (K^n + x^n), where K^n = Hill constant
What happens when the the Hill coefficient (n) is increased?
Binding co-operativity changes, resulting in ultrasensitivity
If n > 1, responsiveness is very large
What is the equation for input signal activating?
f(x) = k’ + k(x^n / (K^n + x^n))
What is the equation for output signal activating?
f(x) = k’ + k(K^n / (K^n + x^n))
What does k and k’ represent in response curve graphs?
k’ = basal rate of expression (independent of TF)
k = maximal additional rate achieved through activation (activation via ligand)
What occurs in digital logic?
A component switches with no intermediate state between two clearly defined states (ON or OFF)
What are analogue responses?
Graded responses to an input signal, so that the output progressively changes as a function of the input signal (concentration)
Which behaviour occurs more commonly in nature, digital or analogue?
Analogue (e.g. can be utilised to construct a gain-tunable transcriptional amplifier)
Digital type can be engineered using biological components
How can the challenge of circuit-host interactions be addressed?
If you build a system that produces a toxic product/will be a burden on the host cell - have it be regulated so that the cell can grow normally (control on and off)
How is gene expression noise generated and what does it result in?
Random encounters generate rate fluctuations
Results in cell-to-cell fluctuations in protein
numbers
How can transcriptional noise be measured?
smFISH
Noise = standard deviation (σ) / Mean mRNA/cell (µ)
What causes an increase in transcriptional noise?
Noise increases with decreasing promoter strength
What may automation of circuits accelerate and what does it require?
Automation may accelerate construction of synthetic circuitry but does require ‘isolation’ and standardisation of the components if behaviour of the biological circuits is to be predictable
What are the applications of synthetic biology in engineering?
Biosynthetic pathways in microorganisms to make (white) biotech products (‘green’ tyre maufacturing)
Biosynthetic pathways in microorganisms to make drugs (anti-malarial drugs)
Plant-based pathways and products
Removing industrial contaminants from the environment (biocides, toxic chemicals, heavy metal contaminants)
Biosensors (arsenic biosensor)
Optogenetics in mammalian systems
What are two examples of plant synthetic biology?
Three Erwinia genes under tuber-specific promoter control catalyse conversion of GGPP to β-carotene and other carotenoids - gold-coloured potatoes
Red light converts Phy B into the active far-red form that heterodimerises with PIF6. This switches on transcription of a reporter gene in tobacco protoplasts. Far-red light turns the system off.
How to build an arsenic biosensor?
arsR encodes a metalloregulatory repressor protein. As binds to ArsR and reduces the repressor’s affinity to the arsR promoter. Coupling the latter to a reporter gene creates a sensitive and inexpensive biosensor
