Coding

Part:BBa_K526002

Designed by: Sung Kuk Lee   Group: iGEM11_UNIST_Korea   (2011-09-26)

Temperature dependent Ribo-Switch fused to GFP

Previosuly, Olga et al (2011) described a temperature dependent riboswitch involved in regulating bacterial chemotaxis at high temperature (Fig 1A). They reported that the mRNA carrying the riboswitch was stable at all temperatures however, the protein expression was favored only at low temperature indicating a robustness in regulating bacterial chemotaxis (Fig 1B and Fig 1C).

SKL2.jpg

We believe that this low-temperature dependent riboswitch would be of great interest in heterologous protein expression. Favoring expression only at a low temperature would gurantee that the heterologous proteins are folded properly without the formation of inclusion bodies.


We have exploited the temperature dependent riboswitch that is associated with the chemotaxis response regulator, CheR, to develop a novel temperature regulated gene expression system. The mRNA is assumed to form a stable secondary structure burrying the RBS and thus blocking gene expression at a higher temperature (37 degree C) where as the temperature is lowered to about 30 to 27 degree C the stability of the secondary structure is disturbed thus exposing the RBS and favoring gene expression.



SKL1.jpg

In 2011 iGEM, we tried to engineer low temperature sensor into E. coli. As is shown in the above figure, when GFP was fused with a temperature dependent riboswitch (Fig 2B) it could sense low temperature: There was no GFP expression at 37 degree celsius. But a significant GFP expression was found at 30 degree celsius indicating the functionality of the ribo-switch.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 630
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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