Team:CU-Boulder

Certain organisms use bioluminescence for communication, mating, or intimidating predators. Our project aims to harness the bioluminescence genes, known as the Lux operon, from Aliivibrio fischeri, to make a genetically engineered E. Coli machine. The six genes of the 9-kilobase Lux operon, LuxCDABE and G, are regulated by LuxI and LuxR and produce blue-green light through the following reaction:

FMNH2+O2+R-CHO → FMN + R-COOH + H2O + Light

Instead of simply transforming an E.Coli with the Lux operon under a single promoter, we have isolated each of the six genes independently and placed them under different promoters to optimize the system and produce the maximum amount of light. The entire system is regulated by a light-repressed promoter so the bacteria only luminesce during nighttime. In conclusion, we have created the first ever bacteria night light! Possible real world applications for this are endless and include illuminating street signs, marking hiking trails, and even giving light to impoverished areas of the world that do not have access to electricity.

Our project harnesses the use of quorum sensing to prevent bacteria from activating pathogenic factors triggered by AHLs. We developed a model system with Allivibrio fisheri. A. fisheri luminesces when activated by AHLs, which is a much safer alternative to pathogenic bacteria. We grew up our E. coli with Vibrio fisheri, and showed that the recombinant E. coli could detect V. fisheri’s AHLs, and inhibit V. fisheri from activating its quorum response genes by not producing light.The detection system then secretes factors that reduce the concentration of AHLs in the environment, thereby inhibiting the bacteria that use AHLs from communicating with each other. This is useful in preventing pathology from bacteria. When a threshold level of AHLs is reached in pathogenic bacteria, the signals activate transcription of pathogenic factors such as biofilm formation and host invasion signals (Raffa et al, 2005). We propose that disrupting the AHLs will inhibit these bacteria from creating biofilms or activating their pathogenic genes. In order to detect AHLs, we took advantage of the Salmonella enterica serovar typhimurium AHL receptor SdiA, a LuxR homolog. The SdiA transcription factor has been shown to be more sensitive to a greater diversity of AHLs than LuxR (Smith et al, 2008). Therefore it will be activated by AHLs present before the AHLs can activate genes in the surrounding bacteria. We also synthesized LuxR mutants that were more sensitive than LuxR at detecting the AHL 3OC6HSL, so they would be activated before LuxR gets activated. We planned to couple the new LuxR homologs to the AHLase Aiia, to demonstrate how we could inhibit activation of pathological factors. We also designed a construct with the new part, NucB, a nuclease that has been shown to digest extracellular DNA.