Experimental Design
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.
Experimental Design
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.
PUF-PIN Fusion Proteins
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 *PUF-PIN) our main source of experimental data.
The light blue symbol labeled *PUF-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 only a single base pair difference between the 8-base pair PUF-PIN and *PUF-PIN RNA recognition sites. Otherwise, their endonuclease function is theoretically unaffected by the different recognition subunits.
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