From 2007.igem.org

Constructing the Reporter Plasmid

The Original Plan:

To test our model experimentally, we need to develop a biological system that will determine if a cell goes lytic or lysogenic after infection. Our construct must contain three important elements:

1) a bistable promoter

2) Green fluorescent protein (GFP)

3) Yellow fluorescent protein (YFP)

The bistable promoter will determine if the cell goes lytic or lysogenic after infection. If the cell goes lytic, enough Cro is produced to fluoresce the cell green. If the cell goes lysogenic, enough CI is produced to fluoresce the cell yellow. Because a bistable promoter was used, we need to first “flip” GFP.

While trying to “flip” GFP we came across a few technical difficulties. It was believed that YFP had possibly been mislabeled or contaminated. After having similar problems with other registry parts, our lab decided to undertake a sequencing project of the entire registry. We believe that this will help with quality control and help eliminate future problems.

Alternative Plan: After having difficulties creating the construct, we decided it would be in our best interest to synthesis the construct. The synthesized construct contained commercial parts, dsRED and acGFP instead of YFP and GFP.