IGEM:IMPERIAL/2009/Encapsulation/Parts/Module3

Inducible promoter

Trigger

Problem

As the encapsulated cell is impermeable/not very receptive to solutes, we need physical means of induction

Possible Solutions

This could be solved in the following ways:

• light
• temperature
• nutrient deprivation.

For temperature, it could be regulated using the following mechanisms:

• Temperature sensitive lambda cI repressor
• RNA thermometer
• Cold sensor hdbB

Our solution

Using the temperature sensitive lambda cI repressor

BBa_K098995 by Harvard 2008 (on our 2009 distribution plate)
http://partsregistry.org/Part:BBa_K098995

This is a thermosensitive cI (clts) inducible system driven by a strong promoter.

For strong promoter J23114
Variant RFP= 256 au

BBa_R0051 is a standard lambda cI regulated promoter.

This system is leaky so we have decided to make our own or obtain a commercially available thermoinducicble system.

Restriction Enzymes-Methylase system

Restriction Enzymes

Dpn1

• blunt cutter
• recognises GATC

Taq1

• staggered cutter
• recognises TCGA

Weak constitutive promoter

To maintain a basal level of methylation

Using BBa_J23103
variant RFP= 17 au
A weak promoter in the popular constitutive promoter family.
http://partsregistry.org/Part:BBa_J23103

Overall design

The schematic below shows the overall design of the M3 system:

Useful reading

| Harvard Thermoinducible cI system

1. Heinemann SF and Spiegelman WG. Control of transcription of the repressor gene in bacteriophage lambda. Proc Natl Acad Sci U S A. 1970 Nov;67(3):1122-9. DOI:10.1073/pnas.67.3.1122 |

2. Gardner TS, Cantor CR, and Collins JJ. Construction of a genetic toggle switch in Escherichia coli. Nature. 2000 Jan 20;403(6767):339-42. DOI:10.1038/35002131 |