Engineering a Light Activated Caspase-3 for Cell Biology Research and Cancer Treatment

Optogenetics is the study of how light can be used to control specific processes in living cells by hinging microbial proteins, which respond to visible light, to model animals. My project was to create a red-light activated caspase-3. Caspase-3 is the final protease that initiates apoptosis (cell death). To engineer this light activated caspase-3, a red-light sensing photoreceptor was fused to a caspase-3 mutant. This gene was inserted into a plasmid and transformed into Escherichia coli. Active caspase-3 cleaves an essential gene in E. coli, causing the bacteria to die. The goal was to have the caspase-3 be active in red-light and inactive in the dark. Eight fusion constructs were created. Seven out of eight showed caspase-3 activity was high (cells died) when grown in the dark and low (cells lived) when grown in the red-light. These results are the opposite of our objective, but show that a light-controlled caspase-3 can be made. Further creation of fusion constructs may identify a light activated caspase-3. A red-light activated caspase-3 could have great significance. The ability to control the death of a single cell would create new opportunities to study the impact a cell has on an organism and possibly treat diseases.