PUF Results Overview
The results covered in this section are of the experiments overviewed in the Design section. Please note that not all planned experiments have been completed or have data ready for disclosure. The data that is complete, that which has been collected and compiled, is listed to the left for their respective experiments.
Results Overview
The results covered in this section are of the experiments overviewed in the Design section. Please note that not all planned experiments have been completed or have data ready for disclosure. The data that is complete, that which has been collected and compiled, is listed to the left for their respective experiments.
Mutant PUF Expression Gel
This is our PUF protein expression gel. We decided to test the PUF protein expression under different conditions to optimize it our further experiments. Lanes labeled 2 through 7 were expressed in a higher, E. Coli standard temperature of 37 degrees C. Lanes 8 through 13 were conducted in a lower temperature of 24 degrees C. The lower temperature, as suggested by the Zefang Wang Lab at the University of North Carolina, has been confirmed to result in more specific and definite PUF protein expression in DH5a.
The parts highlighted in red are significant expressions of the wild type PUF protein.
YFP Fluorescence Data
With regards to our hypothetical results, our experimental fluorescence measurements were substantially faithful to our predictions. Looking at columns 5, 6, 7, and 8, our observations appear to be in line with what we have predicted in introducing the wild type PUF-PIN to constructs containing specific recognition site. However, YFP expression was silenced regardless of the binding site type present in the YFP expression constructs. With many control experiments and repeated tests, we thought the most possible reason could be that PUF recognizes sequences in the YFP gene (although the YFP sequences provided from the registry showed no PUF binding sites
in silico). Columns 1 and 2 are negative fluorescence controls while Columns 3, 4, 5, and 6 are positive fluorescence controls.
As a reference to our aformentioned experimental design predictions. The theoretical results chart is shown here again.
mCherry Fluorescence Data
In order to facilitate more reliable results, we also tested our constructs with the expression of an RFP, mCherry. If in the case that confounding factors influenced our results in our experiments using YFP, we could minimize inaccurate influences on our data by using a different reporter.
As of now, we are still running more mcherry experiments. The data showed some preliminary results.
The results confirmed our hypothesis that PUF-PIN don’t silence the gene from the control construct. This confirms our hypothesis that PUF-PIN doesn't silence the gene from the control construct.
Conclusion
The YFP fluorescence data has shown that the PUF-PIN domain has the ability to silence YFP expression. However, YFP expression was silenced regardless of the binding site type present in the YFP expression constructs. Assessing the specificity of PUF requires further experimental research. The mCherry data suggests that it is likely due to a problem with the YFP reporter, but due to the fact that there was no control binding site cloned into the mCherry expression construct, more experiments will be required for conclusive evidence. The specificity and customizability of our gene silencing through RNA scission both have extensive ongoing experiments.
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