Karen Polizzi Flashcards
Define Part
Piece of DNA that performs a particular function
Define Device
Assembly of parts that performs a user defined function
Define System
Multiple Standard devices, performs a high level function
Methods of changing effective rate of synthesis:
- 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
Potential effects of changing the DNA sequence of a promoter:
Affects the ability of:
- Repressor/Activator binding
- Polymerase recruitment
- Basal levels of transcription
- Dynamic range of transcription output
- Ratio on/off
Define Noise in mRNA transcription
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
Define crosstalk
The ability of other similarly structured inducer molecules to activate gene expression to a similar degree to the gene’s native inducer/ cognate pair
What are the specifications for system of devices to exhibit orthogonality
- > 2-fold activation of gene expression
- < 2-fold crosstalk
Define activator
Increases the rate of RNA polymerase binding
Define inhibitor
Decreases/inhibits the rate of RNA polymerase binding
Draw a DNA plasmid Map
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
2 types of sequences in a promoter
- 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)
Define RBS and how does the sequence surrounding it affect the rate of translation
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)
Describe the method of increasing the rate of protein or mRNA degradation
- Encode for a short terminal amino acid sequence or poly-A-tail
- More limited in tunability than other methods
What are the role of transfer functions in modelling gene expression?
- 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
What does AHL stand for
Acyl Homoserine Lactone
Define Synthetic Biology
- The design and construction of pathways and/or organisms that do not occur naturally
- The redesign of biological systems from the ground up
Technological advances that have made engineering biologial systems possible
- High throughput DNA sequencing and synthesis technologies
- Increased computing power (better tools to integrate and analyse data and to model and simulate
What is the vision of engineering biology
Simplify systems as much as possible and find the subsystems responsible for overall behaviour
Define Standardisation
Ensure data from different labs can be compared by using the same strains (type of cells), protocols and maximising automation of tasks (reducing errors)
Characterisation
Description of part that enables prediction of behaviour - Therefore allows the reuse of standard DNA parts in other designs reliably
Decoupling
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)
Abstraction
Incorporating detailed information about individual coimponents into simpliofied representations of their behaviour
Functional Modularity
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
Define static performance
The characterisation of a DNA part’s normal ‘on’ behaviour
Define dynamic performance
The characterisation of a DNA part’s behaviour after switching
How can characterisation experiments be done?
- 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
