<p class="body-type mainwrap">We finally tested the a plasmid bound BxbI recombinase and a reporter plasmid with an inverted promoter (shown below). The BxbI recombinase should put the promoter in the proper orientation, allowing for deGFP expression. Fluorescence from a reaction containing recombinase and reporter again shows only background levels of fluorescence as compared to a reaction with no DNA. A reaction containing the recombinase and the constitutively active deGFP showed expression at about 66% as compared to a reaction containing just a constitutively active deGFP plasmid. In this case, we believe that the decrease may be caused by transcription and translation machinery is being diverted to produce the recombinase. Future experiments will be aimed at testing additional types of recombinases in cell free and understanding in more detail the mechanisms leading to the decreased deGFP expression. </p>
<p class="body-type mainwrap">We finally tested the a plasmid bound BxbI recombinase and a reporter plasmid with an inverted promoter (shown below). The BxbI recombinase should put the promoter in the proper orientation, allowing for deGFP expression. Fluorescence from a reaction containing recombinase and reporter again shows only background levels of fluorescence as compared to a reaction with no DNA. A reaction containing the recombinase and the constitutively active deGFP showed expression at about 66% as compared to a reaction containing just a constitutively active deGFP plasmid. In this case, we believe that the decrease may be caused by transcription and translation machinery is being diverted to produce the recombinase. Future experiments will be aimed at testing additional types of recombinases in cell free and understanding in more detail the mechanisms leading to the decreased deGFP expression. </p>
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
Characterizing Recombinase Activity in Cell Free
Once toehold switches were functional in cell free, we then shifted to generating functional recombinases in our cell free system. First we set out to characterizing the performance of commercially available Cre recombinase in cell free. Using a terminator excision reporter plasmid (shown below), the Cre recombinase should excise the premature termination and allow for deGFP expression. When fluorescence from this reporter plasmid with added Cre recombinase is compared to fluorescence from a reaction with no DNA and a reaction with constitutively active deGFP, only background levels of fluorescence are seen from the reporter. In addition, to see how the Cre recombinase affects cell free activity, we set up a reaction with Cre recombinase and the constitutively active deGFP plasmid. In this case, we see that the fluorescence is only 25% of the fluorescence shown with the fluorescence. We believed that this may have been a result of the solution containing the Cre recombinase. Our next step was to determine if similar results occurred when testing with plasmid contained recombinases.
FIGURE
We finally tested the a plasmid bound BxbI recombinase and a reporter plasmid with an inverted promoter (shown below). The BxbI recombinase should put the promoter in the proper orientation, allowing for deGFP expression. Fluorescence from a reaction containing recombinase and reporter again shows only background levels of fluorescence as compared to a reaction with no DNA. A reaction containing the recombinase and the constitutively active deGFP showed expression at about 66% as compared to a reaction containing just a constitutively active deGFP plasmid. In this case, we believe that the decrease may be caused by transcription and translation machinery is being diverted to produce the recombinase. Future experiments will be aimed at testing additional types of recombinases in cell free and understanding in more detail the mechanisms leading to the decreased deGFP expression.
