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Team:UIUC-Illinois/Project/Design
From 2012.igem.org
In designing our project we based our quantitative tests on fluorescence measured by a plate fluorescence reader. Our constructs were created in ways that best suit providing evidence for our hypothesis of the PUF-PIN fusion protein showing endonuclease activity. Generally, our results were collected from quantifying two main PUF-PIN fusion protein types, a wild type and a mutant type, in different conditions. These two fusion proteins had recognition sites that differed by two base pairs.
Click on the list to the left to read about each of our constructs and why we decided to do them. All expressions were done in vivo with the DH5a strain of E.Coli on pBAD30 or pPROTet.E plasmids.
In designing our project we based our quantitative tests on fluorescence measured by a plate fluorescence reader. Our constructs were created in ways that best suit providing evidence for our hypothesis of the PUF-PIN fusion protein showing endonuclease activity. Generally, our results were collected from quantifying two main PUF-PIN fusion protein types, a wild type and a mutant type, in different conditions. These two fusion proteins had recognition sites that differed by one base pair.
Click on the list to the left to read about each of our constructs and why we decided to do them. All expressions were done in vivo with the DH5a strain of E.Coli on pBAD30 or Protet plasmids.
The blue symbol labeled PUF-PIN represents the gene that is expressed to produce a wild-type PUF fused to a PIN endonuclease. The comparison of this construct's results to the mutant PUF-PIN (labeled mPUF-PIN) our main source of experimental data.
The light blue symbol labeled mPUF-PIN represents the gene that is expressed to produce a mutant-type PUF fused to a PIN endonuclease. The variation between our two constructs is a two base pair difference between the 8-base pair PUF-PIN and mPUF-PIN RNA recognition sites. Otherwise, their endonuclease function is theoretically unaffected by the different recognition subunits.
Fig. 1 and 2 include the resulting PUF-PIN and mPUF-PIN proteins interacting with their respective binding sites. The wild type binding site is represented by the orange oval. The mutant type binding site is represented by the light orange square. The yellow rounded rectangle represents the YFP reporter gene. As the PUF-PIN fusion proteins, wild and mutant, interact with their respective sites, cleavage activity occurs due to the fused endonuclease.
Fig. 3 and 4 depict the aformentioned endonuclease activity due to the endonuclease, PIN, fused to both PUF and mPUF. After the PUF-PIN complexes recognize and bind to their encoded sites, the endonuclease begins to cut the third base within the 8 bases counted from 5'-3' ; the fourth base downstream the 8 bases counted from 5'-3'.
Since our YFP reporter gene is located downstream of the recognized PUF-PIN cut site, we would be able to quantify changes in expression and fluorescence with and without the introduction of our specific RNA binding endonuclease activity. We hypothesized that in measuring the fluorescence levels, we would have evidence supporting PUF-PIN and mPUF-PIN as RNA scissors with the ability to silence genes.
In order to test whether the endonuclease activity is specific to the PUF binding site, we propose to match both the PUF-PIN and mPUF-PIN proteins with a controlled wild/mutant binding site with a YFP reporter. The control YFP reporter construct encodes a binding site that doesnt recognize both wtPUF/mPUF protein.
In order to eliminate the possibility of confounding factors affecting our apparent results, PUF-PIN experimental results were further reinforced with tests designed to consider other factors of our constructs possibly affecting our quantitative and qualitative measurements. Along with basic experiments testing our proposed constructs we also tested several controls relative to each of the different pieces of our constructs including the following:
| Experiment | Purpose |
| DH5a E. Coli alone | Measure the base fluorescence of DH5a and ensure our strain does not have contaminating fluorescence. |
| Protet plasmid | Measure the base fluorescence of a plasmid we used. |
| YFP + Control Binding Site in protet plasmid | This is a positive control for the YFP experiments. |
| YFP + Specific Binding Site in protet plasmid | This is a positive control for the YFP experiments. |
| YFP + Control Binding Site in protet plasmid + pBAD30 Plasmid | This can help understand whether cotransformation of two plasmids in the cell will interfere with YFP expression. |
| YFP + Specific Binding Site in protet plasmid + pBAD30 Plasmid | This can help understand whether cotransformation of two plasmids in the cell will interfere with YFP expression. |
| YFP + Specific Binding Site in protet plasmid + wild type PUF-PIN in pBAD30 plasmid | This is a theoretically negative fluorescence control due to the endonuclease activity of a specifically bound PUF-PIN protein silencing the YFP gene. |
| YFP + Control Binding Site in protet plasmid + wild type PUF-PIN in pBAD30 plasmid | This is a theoretically positive fluorescence control due to the endonuclease activity of a Control Binding Site bound PUF-PIN protein silencing the YFP gene. |
| mCherry + Control Binding Site in protet plasmid | This is a positive control for the mCherry experiment |
| mCherry + Wild type Binding Site in protet plasmid | This is a positive control for the mCherry experiment. |
| mCherry + Mutant Binding Site in protet plasmid | This is a positive control for the mCherry experiment. |
| mCherry + Control Binding Site in Protet plasmid + wild type PUF-PIN in pBAD30 plasmid | This test was to test the effects of the PUF-PIN endonuclease activity on a reporter other than YFP to pinpoint possible problems stemming from a YFP reporter. Theoretically, we will see fluorescence. |
| mCherry + Control Binding Site in Protet plasmid+ mutant PUF-PIN in pBAD30 plasmid | This test is to show the specificity of mutant PUF-PIN. Theoretically, we will see fluorescence. |
| mCherry + Specific Binding Site in Protet plasmid+ wild type PUF-PIN in pBAD30 plasmid | This test was to test the effects of the PUF-PIN endonuclease activity on a reporter other than YFP to pinpoint possible problems stemming from a YFP reporter |
| mCherry + Specific Binding Site in Protet plasmid+ mutant PUF-PIN in pBAD30 plasmid | This test is to show mutant PUF-PIN's functionality. Theoretically, no fluorescence will be observed. |
The above chart shows our predicted results. The control binding sites are predicted to bind both wild and mutant PUF-PIN fusion proteins. The only non-expression combinations are when we match a PUF-PIN or mPUF-PIN to it's respective binding site which should result in endonuclease activity and subsequent silencing of the YFP reporter gene.
