Difference between revisions of "Team:BostonU/Results"

Characterizing Our Cell Free System

The first step in accomplishing our project was to characterize the activity of the cell free we made in house. This required measuring the maximum expression levels the can be accomplished in the cell free system. We did this by modelling the saturation of fluorescence from the system. The results of the model can be found on our modelling page. We measured the expression from varying concentrations of a plasmid coding for a constitutively active deGFP gene. We found that maximal expression in the cell free system is achieved around 20nM of DNA.

Characterizing Toehold Switch Activity in Cell Free

The next step in validating the functionality of our project was to characterize toehold switch functionality in cell free. The best expression was achieved when activating the toehold switch with RNA trigger at 10,000X concentrations relative to the toehold. In this experiment, fluorescence from reactions with plasmid toeholds and RNA trigger was compared to fluorescence from reactions containing plasmid toehold switches alone, no DNA at all, and a plasmid containing a constitutively active deGFP. Measurable fluorescence is achieved from two different toehold switch/trigger pairs, but the expression is lower than that seen from the constitutively active deGFP. Larger amounts of RNA may allow for better fluorescence, but the methods we employed for transcribing the trigger DNA to RNA was not able to produce higher concentrations.

FIGURE