Module Integration - Thermoinduction

Module 2: Encapsulation

Overview

When sufficient protective coating has been made, the temperature of the system is raised to 42°C. This activates the thermosensitive promoter, consequently triggering module 3.

Rationale

After encapsulation, it becomes more difficult for normal chemical induction to penetrate the outer protective coating. To tackle this problem, heat induction is used. Heat induction is, in addition, a commonly used and highly reproducible induction method in industrial scale manufacturing processes.

The thermosensitive cI promoter is chosen as it is widely cited in literature for efficient thermoinduction of systems [1], and has been proven to work well in commonly available plasmids. [http://partsregistry.org/Part:BBa_K098995| BBa_K098995], in particular, is the only construct for the thermosensitive cI system in the registry and has already been characterised previously.

The system

The lambda promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_R0051| R0051]) is strongly repressed at 28 degrees by the cI repressor([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098997| K098997]). Therefore, at 28 degrees, the thermoinducible system has no activity.

However, this cI repressor is unique in that it is temperature-sensitive, and will readily lose its functional structure as the temperature increases. Therefore, when the temperature is raised to 42 degrees, the lambda promoter is no longer repressed and can trigger module 3.

  About the thermoinduction system

Characterisation of existing biobrick

The thermoinducible construct ([http://partsregistry.org/Part:BBa_K098995| BBa_K098995]), an existing biobrick, is characterised by ligating it to GFP. The cells are grown at the low temperature of 28 degrees where the promoter is inactive. The temperature is then shifted up to 42 degrees. The GFP fluorescence is monitered throughout as an indicator of promoter activity.


The GFP fluorescence is shown to be at baseline when cells are grown at 28 degrees. However, when the cells undergo a temperature shift to 42 degrees, there is an almost immediate increase in GFP fluorescence.

This shows that the activity of the lambda promoter does increase when the temperature is shifted up, so the thermoinducible construct ([http://partsregistry.org/Part:BBa_K098995| BBa_K098995]) does become activated by higher temperatures.


  About our wetlab results

References

1. Wolfgang Jechlingera*, Michael P Szostaka 1 , Angela Wittea, Werner Lubitza (1999) Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene E expression in Escherichia coli. FEMS Microbiology Letters. 173(2), pp 347 - 352