Difference between revisions of "Part:BBa K779504"

Latest revision as of 21:13, 8 October 2012

Long scrambled input strand MammoBlock

Long non-matching (scrambled) input strand to act as a negative control to the reporter formed by parts Part:BBa K779500 and Part:BBa K779502 or Part:BBa K779501 and Part:BBa K779502. Since this strand has the correct toehold it can bind to the exposed toehold of the reporter, but since this input has the incorrect hybridization domain, it can not displace the top strand of the reporter. Thus this scrambled input is a negative control to strand displacement. Refer to the referenced part pages for more information.

Has an infrared marker on 5' end for quantification of oligo levels.

Modifications: 5' IRD800 fluorophore, 3' phosphate.

IDT DNA Oligo Order: /5IRD800/mAmCmUmAmAmCmCmUmAmAmCmUmAmCmAmAmCmAmAmCmAmCmUmAmUmCmCmA/3Phos/

Transfection data showing that the scrambled input has no effect on a strand displacement reaction:

This graph shows flow cytometry data with FITC on the x-axis in A.U. and Texas-Red on the y-axis. A 200-point moving average was taken to reduce noise in the data. We can see that for higher levels of green fluorescence (indicating transfection efficiency) in the FITC channel, the no input well and the scrambled input wells have consistent low levels of red fluorescence (indicating no strand displacement). However, for the input well, higher levels of green fluorescence correlate with higher levels of red fluorescence, which indicates that for higher transfection efficiency of our reporter, we see more strand displacement taking place.

Experimental Design: This foundational experiment has finally shown that we can demonstrate RNA strand displacement in vivo after reformulating a new design for our sequences and testing multiple iterations of the design. The experiment also shows that a strand displacement negative control, BBa_K779504 along with the annealed reporter BBa_K779501 + BBa_K779502, indeed does not result in any strand displacement. In this experiment, each well received 150,000 HEK293 cells in supplemented DMEM. For controls, No Transfection is a well which only received cells. The No Input well was transfected with 25 pmol of our new designed longer reporter BBa_K779501 annealed to BBa_K779502 using RNAiMAX reagent. The experimental well labeled Scrambled Input, was transfected with 25 pmol of both the reporter and an input strand with the correct toehold but incorrect binding domain (BBa_K779504), which would not yield a strand displacement reaction. The experimental well labeled Input, was transfected with 25 pmol of both the reporter and the input strand with the correct toehold and binding domain (BBa_K779503), which can bind to the exposed toehold of the reporter and through branch migration, knock off the top strand of the reporter, yielding an unquenched fluorophore and successful strand displacement reaction In Vivo.

Usage and Biology

Used as a negative control for strand displacement reactions. See referenced parts above for more information.

Sequence and Features