Composite

Part:BBa_K2836002

Designed by: Chang Wei-Tse   Group: iGEM18_SHSID_China   (2018-10-07)


luxCDABEG-luxG

This bacterial lux operon (luxCDABEG)encodes for the endogenous reactants needed in a light-emitting chemical reaction in the bioluminescent bacteria Aliivibrio fischeri. The luxA and luxB genes code for the alpha and beta subunits of the bacterial luciferase, luxC, luxD, and luxE genes encode for the r,s, and t polypeptides of the fatty-acid reductase complex that synthesizes and recycles the aldehyde substrate for luciferase, and luxG codes for the flavin reductase needed in regenerating the reduced flavin mononucleotide used in the reaction. As the flavin reductase has been shown to increase light production in bacteria, the 2018 SHSID_China iGEM team has synthesized and attached another luxG gene from Aliivibrio fischeri strain ES114 onto the original lux operon to increase light output.

In order to show that the additional luxG gene increases the intensity of light produced by biochemical reaction encoded by the lux operon, experiments were conducted to investigate the difference in light production between part BBa_K2836002 (abbreviated 2G) and part BBa_K325909 (abbreviated 4L). 30 test tubes of E. coli were transformed with each part and grown (giving a total of 60 test tubes) in a 37 degrees Celsius incubator shaker overnight. Arabinose was added to make the final concentration in each test tube 0.1M, and luminescent and photometric measurements were taken using a Thermo Scientific Varioskan Flash 4.00.53 every hour over a course of five hours. Each data point had 6 repeats and the average was taken to ensure the accuracy of the measurements and negate the effects of outliers. The raw data table is shown below. T--SHSID_China--partpage1.png

We processed our data and acquired our numbers for the relative luminescence of the bacteria by dividing the average Abs by the Abs at 0Hrs, dividing the average luminescence by the luminescence at 0Hrs and taking the ratio of the two for each of the parts at a specific time. The formula is as thus:(Average Luminescence/0Hrs Luminescence)/(Average Abs/0Hrs Abs). Then, we plotted the graphs of the relative luminescence of the two parts as shown below. 800px-T--SHSID_China--partpage2.png

From the graph, we can determine that an additional luxG does have an impact on the light emission of bioluminescent bacteria, and that part BBa_K2836002 is a functional improvement over part BBa_K325909 in terms of light intensity.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
    Illegal NheI site found at 4171
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3169
    Illegal BamHI site found at 1144
    Illegal XhoI site found at 3999
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 5558
    Illegal BsaI.rc site found at 2567
    Illegal SapI.rc site found at 5883
    Illegal SapI site found at 961


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