Karen Polizzi

Question 1

Define Part

Answer 1

Piece of DNA that performs a particular function

Question 2

Define Device

Answer 2

Assembly of parts that performs a user defined function

Question 3

Define System

Answer 3

Multiple Standard devices, performs a high level function

Question 4

Methods of changing effective rate of synthesis:

Answer 4

• Increase or decrease the number of DNA copies
• Increase or decrease the rate at which mRNA is made
• Increase or decrease the rate at which the protein is translated
• Change the rate of degradation of mRNA or Protein

Question 5

Potential effects of changing the DNA sequence of a promoter:

Answer 5

Affects the ability of:

• Repressor/Activator binding
• Polymerase recruitment
• Basal levels of transcription
• Dynamic range of transcription output
• Ratio on/off

Question 6

Define Noise in mRNA transcription

Answer 6

As there are small numbers of molecules in cells, the rate of transcription will occur in bursts (as the RNA polymerase is more likely to return to the start after it has encoded the gene). This generates stochastic effects of the model called noise

Question 7

Define crosstalk

Answer 7

The ability of other similarly structured inducer molecules to activate gene expression to a similar degree to the gene’s native inducer/ cognate pair

Question 8

What are the specifications for system of devices to exhibit orthogonality

Answer 8

• > 2-fold activation of gene expression

- < 2-fold crosstalk

Question 9

Define activator

Answer 9

Increases the rate of RNA polymerase binding

Question 10

Define inhibitor

Answer 10

Decreases/inhibits the rate of RNA polymerase binding

Question 11

Draw a DNA plasmid Map

Answer 11

In rotational Order:

Close together:

• Promoter
• 5’ Primer Site (Short)
• Restriction Site (Really Short)
• Inserted Gene (Decent Chunk)
• Restriction Site
• 3’ Primer Site
• Origin of Replication
• Antibiotic Resistant Gene
• Selectable Marker

Question 12

2 types of sequences in a promoter

Answer 12

• Core Sequences (essential for polymerse binding)
• Intervening regions (randomising these will affect the ability of the RNA polymerase to bind and so the rate of transcription)

Question 13

Define RBS and how does the sequence surrounding it affect the rate of translation

Answer 13

The sequence upstream of the Ribosome Binding Site can affect the mRNA secondary structure (folding) and so the ability of the ribosome to bind and initiate translation
(There are calculators that calculate dG 30bp folding)

Question 14

Describe the method of increasing the rate of protein or mRNA degradation

Answer 14

• Encode for a short terminal amino acid sequence or poly-A-tail
• More limited in tunability than other methods

Question 15

What are the role of transfer functions in modelling gene expression?

Answer 15

• Aid in transition towards automated design
• Provide a simpler representation of input-output behaviour
• Correlate the concentration of inducer to output of gene expression module
• Most curves are sigmoidal

Question 16

What does AHL stand for

Answer 16

Acyl Homoserine Lactone

Question 17

Define Synthetic Biology

Answer 17

• The design and construction of pathways and/or organisms that do not occur naturally
• The redesign of biological systems from the ground up

Question 18

Technological advances that have made engineering biologial systems possible

Answer 18

• High throughput DNA sequencing and synthesis technologies

- Increased computing power (better tools to integrate and analyse data and to model and simulate

Question 19

What is the vision of engineering biology

Answer 19

Simplify systems as much as possible and find the subsystems responsible for overall behaviour

Question 20

Define Standardisation

Answer 20

Ensure data from different labs can be compared by using the same strains (type of cells), protocols and maximising automation of tasks (reducing errors)

Question 21

Characterisation

Answer 21

Description of part that enables prediction of behaviour - Therefore allows the reuse of standard DNA parts in other designs reliably

Question 22

Decoupling

Answer 22

Detaching systems from one another so that there are fewer and easier problems to solve (separation of high level design taks from lower level specifications and parts fabrications)

Question 23

Abstraction

Answer 23

Incorporating detailed information about individual coimponents into simpliofied representations of their behaviour

Question 24

Functional Modularity

Answer 24

The dividing up of the DNA sequence into parts that perform a particular function which can be assembled into devices and systems to perform a high level user defined function

Question 25

Define static performance

Answer 25

The characterisation of a DNA part’s normal ‘on’ behaviour

Question 26

Define dynamic performance

Answer 26

The characterisation of a DNA part’s behaviour after switching

Question 27

How can characterisation experiments be done?

Answer 27

• Create recombinant plasmid with part of interest combined with others
• Select appropriate output protein that can be measured easily such as GFP
• Transform the plasmid host into a cell
• (Add antibiotic resistant gene)
• Make high throughput measurementsof behaviour using microtitre or flow cytometry