Part:BBa_K2070023

Constitutive ecf16 generator

This part is used to constitutively generate the trigger required for the activation of the Toehold rbs in part BBa_K2070018 to generate GFP. The trigger and toehold together form the Toehold System.

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 is contains a specific complementary sequences on both upstream and downstream of the RBS sequence, forming a hairpin structure that encloses the RBS. 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)

(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 BglII site found at 239
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 434
  
• 1000
  COMPATIBLE WITH RFC[1000]