Part:BBa_K898890:Design
wtPUF+PIN
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1297
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 35
- 1000COMPATIBLE WITH RFC[1000]
Characterization Method
This figure includes the resulting PUF-PIN proteins interacting with the binding sites. The wild type binding site, placed in between RBS and YFP, is represented by the orange oval. The yellow rounded rectangle represents the YFP reporter gene.
YFP Fluorescence
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 a control recognition site and a specific recognition site. Columns 1 and 2 are negative fluorescence controls while Columns 3 and 4 are positive fluorescence controls measured without the influence of any sort of binding site.
RFP Fluorescence
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.
However, this experiment (though not as intensively compared with numerous controls as our YFP tests) has yielded unexpected results. Suggesting key differences in the gene silencing ability of PUF when used with mCherry on a Protet plasmid. Though this is not what we expected, these results are still somewhat inconclusive since we did not test the same construct on a pBAD30 plasmid instead of Protet. pBAD30 is what we used for our YFP + binding site + wild type PUF-PIN fluorescence experiments. Further experimentation is underway.
Wild Type PUF-PIN Protein Purification
1 L culture incubated at 37oC till 0.5 nm optical density after inoculation with 5 mL of overnight. 2 mM IPTG induction for 2 hours. His-Tag Ni-NTA purification, centrifuged with Millipore 30kDa cutoff ultracentrifuge tubes. SDS-PAGE 6% native gel stained with Coomassie Brilliant Blue for 1 hr. and destained with H20 for 1 hr.
Endonuclease Assay
Wild type PUF-PIN fusion bound and cleaved RNA as expected.
Source
The PUF domain from human Pum1 (residue 828-1176) and the PIN domain from human Smg6 (residue 1238-1421)
References
Professor Zefeng Wang, University of North Carolina- Chapel Hill
http://www.med.unc.edu/pharm/people/primaryfaculty/zefeng-wang/wang