IGEM:IMPERIAL/2009/Encapsulation/Timers

Timers

Timer Prototype 1.1:

Overview: This timer works on a double repression mechanism. Varying IPTG concentration provides a variable threshold which must be overcome by the accumulation of LacI for the switch to flip. The production of LacI will ultimately be linked to the accumulation of protein.

1) Initial state: LacI=OFF, CI repressor = ON, Output = OFF

2) LacI accumulates until it saturates the IPTG.

3) Once the IPTG is saturated, LacI represses the LacI responsive promoter. This decreases the expression of TetR repressor.

4) A fall in TetR repressor levels results in de-repression of the TetR responsive promoter.

5) Final state: LacI = ON, TetR repressor = OFF, Output = ON

Device Assembly:

BBa_K082025 consists of an RBS fused to LacI followed by a double terminator. This part works although it does not appear to be present in the 2009 starter kit.

Testing Timer Prototype 1.1:

Testing construct 1:

Overview:

Treat the timer module as a black box. Use a POPs input by placing a characterised promoter upstream of RBS2. Obtain a system output by placing a GFP reporter down stream of the TetR promoter.

Input:

To characterise the timer, it must be possible to measure output as function of time. To achieve this, input must be inducible. The device F2621 is well studied and would be suitable for timer characterisation.

Output:

BBa_E0240 takes a POPs input and gives a measurable output of GFP.

Tuning:

Once the transfer function has been elucidated, it will be possible to investigate timer tuning via the addition of IPTG.

Testing construct 1:

This testing construct only requires 2 ligation steps:

Other Timer Info

Interesting and helpful paper on a simple timer [1]

Timers have many biological and engineering appplications. Design of mechanisms and characterisation of parts will provide a reusable timer module. We could have a timer module as part of Modules 1, 2, 3 or 5 of our project.
Previous iGEM teams have explored different genetic circuits required to produce a timer.

Considerations for our timer:

• Separate timer for each phase vs. one timer that is ‘continuous’ between phases
  
• Thresholds
  
• Periodicity
  
• Start/stop vs. oscillation vs. combination of both
  
• Reset function
  
• Pre-programmed function(s)
  

• James Chappell 05:34, 21 July 2009 (EDT):What would the specs for our timer be? You should try to identify these first then look at the existing timers and see if they meet the specs, if not then look for new designs. Also Might be good to add diagrams of how the circuits work.
  

Specifications for a Timer in Each Phase of our Project

Module 1 - Compound:

1. Induce production of compound at a specific time? - eg in response to something.
   

Module 2 - Encapsulation:

1. Induce sporulation/encapsulation once desired threshold of compound X has been produced.
   

Module 3 - Killing Strategy:

1. Induce death after encapsulation.
   

• Have a preset timer that induces death once the bacterium is encapsulated? (Chiba'08)
• Induce death immediately - would a timer be required? Why not just use a promoter? We could set a threshold rather than using a timer mechanism.
  

Module 5 - Delivery

Previous iGEM Timers

Chiba2008

Linear accumulation of non-degradeable signal molecules over time, which are then detected by receivers; in turn activating the switch to an 'on' state.

KULeuven2008

KULeuven used the 'InverTimer' in their 'Dr. Coli' project to induce destruction of their bacteria once the time lapsing between disease signals had reached a sufficient period.

Their system is a LacI based inverter, controlling LuxI production. LuxI produces AHL (quorum-sensing molecule) which in turn gradually activats LuxR and its controlled promoter in the next module/device.

NYMU-Taipei2008

Taipei made two timers based on a three-component system:

Starter, Counter, Stopper

The counter component of the system is an oscillator. They required two timers with different periods so used different oscillators to fit the purpose of each timer.

Cyanoxilator

• A Cyanobacterial oscillator using the Kai proteins in E.coli.
  
• Time period of between 14 - 60 hours (...wide range there??)
  

Reloxilator
'A tuneable intracell-synchronized relaxation oscillator based on the combination of:

• Using the lysogenic genetic swtich from phage lambda and giving it tuneable oscillatory properties.
  
• Using the intracellular synchronising properties of Vibrio fischeri.
  
• Time period of ~46 minutes.
  

The timers are stopped once protein production reaches a pre-set threshold. Once this threshold is reached, the activity of the output promoter changes.

References

1. Ellis T, Wang X, and Collins JJ. Diversity-based, model-guided construction of synthetic gene networks with predicted functions. Nat Biotechnol. 2009 May;27(5):465-71. DOI:10.1038/nbt.1536 |