Part:BBa_K779120

RNA Reporter top strand with quencher (RQ) and tag fluorophore (Alexa 488) MammoBlock

Binds to Part:BBa_K779116 to form a RNA reporter complex (rRep6). Has an unquenched Alexa 488 fluorophore.

Modifications: 5' Iowa Black RQ quencher, 3' Alexa 488 fluorophore, all bases modified with 2'O Methyl (standard modification for increased stability of RNA); ordered as a DNA oligo with Us instead of Ts.

IDT DNA Oligo Order: 5IAbRQ/mCmAmUmAmAmCmAmCmAmAmUmCmAmCmA/3AlexF488N/

Usage

This part works in concert with a complementary strand (Part:BBa_K779116). When the parts are annealed, RQ will quench ROX on the opposite strand, while Alexa continues to fluoresce at all times (Alexa is outside the quenching range of RQ, as by [http://idt.com IDT]).

Part:BBa_K779118 can act as an input to the formed reporter, whereas Part:BBa_K779119 is used as a negative control input. The mechanism by which the input strands react with the reporter, toehold-mediated strand displacement, is described in more detail on the [http://2012.igem.org/Team:MIT MIT iGEM 2012 Wiki page].

Excitation / emission spectrum for the Alexa 488 fluorophore in BBa_K779120. Obtained by the MIT iGEM 2012 team using a Tecan Safire II plate reader (see [http://2012.igem.org/Team:MIT/MaterialsAndMethods#iv2bio instrument settings], note that excitation and emission scans were performed instead of fixed wavelength measurements).

Dilutions of BBa_K779120 as a free strand or in complex with Part:BBa_K779116. Measurements carried out on a Tecan Safire II plate reader (see [http://2012.igem.org/Team:MIT/MaterialsAndMethods#iv2bio protocol]).

In vitro strand displacement reaction using the Part:BBa_K779116:BBa_K779120 complex with input strands Part:BBa_K779118, Part:BBa_K779119 as described above. The results indicate that the Alexa 488 fluorophore does not prevent strand displacement from happening, although it could theoretically inhibit toehold binding and branch migration initiation. Measurements carried out on a Tecan Safire II plate reader (see [http://2012.igem.org/Team:MIT/MaterialsAndMethods#iv2bio protocol]).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]