From 2007.igem.org

Results

Gene Splitting

The negative control (hixC-GFP2) on the left does not fluoresce, but the experimental split GFP (Plac-RBS-GFP1-hixC-GFP2) on the right does.

We were able to split two genes: GFP and RFP. Split GFP has strong green fluorescence. Split RFP's red color is much reduced compared to wild-type RFP. It takes overnight incubation at room temperature for the red color to be visible in white light. Both colors are fluorescent under UV light, although the green color predominates. In the first image below, a negative-control (on the left) does not fluoresce, but split GFP (on the right) does.

We also wanted to test fluorescence phenotypes with both proteins in the same cells. Below on the left are cells containing both split GFP and split RFP, downstream of the T7 RNA polymerase promoter and co-transformed with T7 RNA polymerase plasmid. These cells look bright green, but the red color is hard to see under UV light. Under white light, the green color is not apparent, but the cells are a little pink. The cells only containing split RFP, on the right, are clearly pink under UV light. However, not all cells are equally colored - some are greener than others, which is a curious result as the cells do not contain GFP of any form.

On the left, cells with the T7 RNA polymerase plasmid and a plasmid containing both split RFP and split GFP. These cells fluoresce a bright green under UV light and look pink under white light. On the right, cells with the T7 RNA polymerase and only split RFP demonstrate red fluorescence of varying intensities.

In addition to the regular split GFP and split RFP constructs, we also wanted to test some "hybrid" constructs to ensure that they would not fluoresce. GFP and RFP have similar structures and functions. Previous studies have shown that it is possible to modify GFP to display a wide range of color phenotypes. We created the following parts: Plac-RBS-RFP1-hixC-GFP2 and Plac-RBS-GFP1-hixC-RFP2. A plasmid may, at some point during its flipping process, contain such sequences. We tested to make sure that the similarity of these two proteins did not make them compatible enough to fluoresce. It was found that neither of these parts show any fluorescence or color change.

Constructs

Once we managed to split two genes we proceeded to implement two different graphs in plasmids.

We built the following constructs:

• Graph A
  
• Graph B
  
• Solution to both A and B
  
• Positive Control 1
• Positive Control 2

Part Flipping and Computation

Once we had established the creation of successful split reporter genes it became possible to begin constructing HPP computers.

Cells with only the T7 RNA polymerase plasmid, shown under UV light. There is no fluorescence.

Cells with both the T7 RNA polymerase plasmid and the flipped Hamiltonian Path Problem plasmid, shown under UV light. There is visible green fluorescence.