Part:BBa_K2070007

Toehold Trigger Inducible GFP generator

This part contains a constitutive promoter and a Toehold rbs that requires a specific RNA Trigger sequence to translate the GFP downstream of the rbs. This specific trigger sequence is generated by part BBa_K2070016.

Detailed Explanation of the Toehold System

Ribosomes cannot bind to the Toehold RBS under normal conditions. Therefore, proteins cannot be made because mRNAs cannot be translated even when the gene is transcribed. For a ribosome to bind to a toehold RBS, the existence of a specific short RNA named trigger RNA is necessary.

The previous gene in which the toehold RBS stops transcription becomes activated as shown below when trigger RNA is present, and will be translated. The functioning DNA is represented in yellow.

Why can’t the ribosome bind to the Toehold RBS?

The toehold rbs contains a specific complementary sequences on both upstream and downstream the RBS sequence, forming a hairpin structure that encloses the RBS. The ribosome therefore cannot bind to the toehold RBS and thus downstream genes cannot be translated. This kind of RNA is called switch RNA. However, the hairpin structure of a switch RNA can be broken by what is called the trigger RNA, which includes partially complementary sequence with the switch RNA, making the translation of downstream genes possible.

(Green, A., 2014)

Reference

Green, A., Silver, P., Collins, J., & Yin, P. (2014, 11). Toehold Switches: De-Novo-Designed Regulators of Gene Expression. Cell, 159(4), 925-939. doi:10.1016/j.cell.2014.10.002

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 692
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 709