Download Construction of a genetic toggle switch in

Construction of a genetic toggle
switch in Escherichia coli
Farah and Tom
What is a Toggle Switch?
“A synthetic bistable gene regulatory network”
What is a Toggle Switch?
The switch consists of:
Promoters
Repressors
Inducers
Promoters encourage expression of a gene.
Repressors bind to promoters, inhibiting expression of genes.
Inducers bind to repressors, preventing repressor binding to promoters
and thus encouraging expression.
These components are arranged in a mutually inhibitory fashion.
What is a Toggle Switch?
This model was chosen as it was:
- Simple
- Achieves robust bistable behaviour
How does the toggle switch work?
Promoter 1 encourages expression of Repressor 2
Promoter 2 encourages expression of Repressor 1
Assuming Repressor 2 is in excess of Repressor 1
•
Promoter 1 is not active
•
On introduction of Inducer to Repressor 2
•
Inducer binds repressor 2
•
Repressor 2 can no longer bind Promoter 2
•
Promoter 2 promotes expression of Promoter 1
•
Switching has occurred
The Maths
1
du


u

dt 1  v
2
dv


v

dt 1  u
The Geometric Structure of the
Toggle Equations
pTAK and pIKE
Two classes of toggle switches were constructed:
pTAK
lac repressor represses the Ptrc-2 promoter
Temperature sensitive lambda repressor represses the PLslcon promoter
Induced by: IPTG (lac) or Thermal Pulse (tsLambda)
pIKE
lac repressor represses the Ptrc-2 promoter
tet repressor represses PLtetO-1
Induced by: IPTG (lac) or aTc (tet)
High and Low States
Green Fluorescent Protein (GFP) is used as a reporter.
GFP is under control of Ptrc-2
Ptrc-2
TetR
PLtetO-1
LacI
GFP
Example of a pIKE system with GFP reporter
The state where Ptrc-2 is active, and GFP is being produced is called the High State
The state where Ptrc-2 is repressed, and GFP is not being produced is called the
Low State
Investigating Bistabilty
Six variants were used to investigate the conditions required for bistability
• Four pTAK plasmids
• Two pIKE plasmids
These variants differed by the strength of Ribosome Binding Site 1 (RBS1), where
stronger binding RBS sites lead to greater protein synthesis.
All the pTAK plasmids exhibited bistability and
remained stable for at least 22h.
One of the pIKE plasmids exhibited bistability
The reason for the failure of this pIKE is most likely
because TetR is a weaker repressor than lambda.
Graphs
Switching States
For pTAK117 switching time for low to high state takes around six hours.
Switching States
For pTAK117 switching time for high to low state takes around 30 minutes.
Switching States
Switching from Low to High
This takes upto 6 hours.
This is because IPTG-bound Lac Repressor is gradually diluted by cell growth.
As IPTG-bound Lac Repressor decreases, the amount of TetR produced also
decreases, allowing expression of LacI and GFP
Switching from High to Low
This takes upto 35 minutes.
This is because tsLamdba is immediately destabilised by a temperature increase
Ptrc-2
TetR
PLtetO-1
LacI
GFP
Other potential toggles
In the examples of toggle switches used here the mechanism used to flip the switch
has been introduction of an inducer.
This has limited application in synthetic biology.
There are other ways of achieving the switch which may be of use:
- Addition of extra repressor
- Destruction of repressor (eg. Using UV light)
Applications
Gene Therapy
Biotechnology
Cellular memory unit