1. Additional Characterization of part BBa_K325909

Improving upon the work of the Cambridge iGEM team 2010 and NTNU Trondheim iGEM team 2017, we performed additional experiments to add further information and build upon the characterization of part BBa_K325909. As this part is provided in the 2018 Distribution Kit, we tested the effectiveness of the arabinose induced lux operon-encoded enzymatic pathway for light emission in part BBa_K325909 by transforming it into E. coli, incubating overnight and subjecting it to different final concentrations of arabinose over a course of five hours. Reflecting upon the previous characterizations, we chose a temperature of 29℃ to perform our experiments, since by looking at the information presented by the NTNU Trondheim iGEM team 2017, it seems as if 29℃ may provide us with the brightest results.

Luminometric and photometric measurements were taken with a Thermo Scientific Varioskan Flask 4.00.53 every hour throughout the experiment, and the data was recorded on the raw data table provided below.

The data is analyzed using the formula: (Average Luminescence/0Hrs Luminescence)/(Average Abs/0Hrs Abs) for each concentration of arabinose, and the graphs are plotted below, with part BBa_K325909 abbreviated as 4L:

From the graph, even though the relative luminescence of the bacteria are similar during the first four hours, there is a significant increase in relative luminescence during the fifth hour at a concentration of 0.1M, therefore we have concluded that inducing part BBa_K325909 with 0.1M arabinose will result in the most intense light emission.

2. Construction of the Composite Part BBa_K2836002

To increase the light output from part BBa_K325909, we used de novo synthesis to create an additional luxG gene from a different strain of the same species of bioluminescent bacteria (Aliivibrio fischeri), which codes for the flavin reductase used in reduced flavin mononucleotide turnover, and ligated it onto the existing part to form the composite part BBa_K2836002 with the operon luxCDABEG-luxG under an inducible arabinose promoter.

To prove that the additional luxG gene can increase luminescence, we conducted experimental tests to compare the relative luminescence of the bacteria with each plasmid under a 0.1M concentration of arabinose, which we have determined from previous characterizations of part BBa_K325909 will result in the brightest light. The bacteria were grown in 37℃ overnight before being taken out and put into a 29℃ shaking incubator over the course of the measurements. Part BBa_K325909 is abbreviated 4L and Part BBa_K2836002 is abbreviated 2G.

As shown from the graphs above, when induced with 0.1M arabinose, part BBa_K2836002 consistently showed an higher level of relative luminescence when compared to part BBa_K325909 over the course of five hours, thus affirming the fact that the additional luxG gene does increase light production in bioluminescent bacteria.