Difference between revisions of "Part:BBa K1913014"

Revision as of 07:44, 16 October 2016

3-oxo-hexanoyl-HSL GFP reporter

This part reports (with GFP production) the presence of 3-oxo-hexanoyl-HSL, the autoinducer in the lux quorum sensing system from Vibrio fischeri.

Usage and Biology

3-oxo-hexanoyl-homoserine lactones (HSLs) are known to bind the 'lux-box' in the lux pR promoter (BBa_R0062). Binding of HSL in the promoter induces expression of downstream genes. HSLs function as signal molecules for inter bacterial communication: autoinducers. 3-oxo-hexanoyl-HSL specifically is the autoinducer in the lux quorum sensing system in Vibrio fischeri. This part consists of the lux pR promoter, an RBS and a GFP generator.

We tested the HSL reporter BBa_K1913014 in pSB1C3 with the lux operon part BBa_K546000 in pSB4K5. E. coli DH5alpha cells were transformed with both plasmids (cells containing BBa_K546000 were used to make elctrocompetent cells, which were then transformed with the reporter), these cells were tested overnight in BioTek’s SynergyMx plate reader overnight (figure 1, figure 2).

Figure 1. Fluorescence (excitation: 480nm, emission 510nm, bandwidth: 9nm) and absorbance data for E. coli quorum sensing strains. Error bars indicate standard error of the mean. Continuous line: Fluorescence/OD₆₀₀; Dashed line: Absorbance at 600nm (OD₆₀₀). Orange, Blue: separate biological repeats of the DH5alpha strains containing the 2 plasmids (BBa_K546000 in pSB4K5 and BBa_K1913014 in pSB1C3). Pink: reporter plasmid only strain. For both strains every value displayed is the average of all (4) technical replicates. All Fluorescence/OD₆₀₀ data were normalized to the highest Fluorescence/OD₆₀₀ value measured. After almost 12 hours, the fluorescence from the 2 plasmid strains reached an overflow. Therefore no values after this point are displayed. One biological repeat of the 2 plasmid strain experiment is not shown to improve the clarity of the graph. For completeness, the complete graph is shown underneath here.

Figure 2. Fluorescence and absorbance data for E. coli quorum sensing strains. Error bars indicate standard error of the mean. Continuous line: Fluorescence/OD600; Dashed line: Absorbance at 600nm (OD₆₀₀). Orange, Blue, Green: separate biological repeats of the DH5alpha strains containing the 2 plasmids (BBa_K546000 in pSB4K5 and BBa_K1913014 in pSB1C3). Pink: reporter plasmid only strain. For both strains every value displayed is the average of all (4) technical replicates. All Fluorescence/OD₆₀₀ data were normalized to the highest Fluorescence/OD₆₀₀ value measured. After almost 12 hours, the fluorescence from the 2 plasmid strains reached an overflow. Therefore no values after this point are displayed. One biological repeat (green) showed a high variance between technical repeats in both OD₆₀₀ and Fluorescence/OD₆₀₀ values.

These data show that BBa_K1913014 and BBa_K546000 functions as expected together. When HSL levels are visualized with GFP activity, we see that when cell density (here measured as OD₆₀₀) increases, there is a sharp increase in HSL concentration in the cells. Though a slight increase in fluorescence is also seen in the absence of the quorum sensing genes from BBa_K546000, this increase does not compare to the increase seen in the presence of the lux quorum sensing system. We therefore conclude that BBa_K1913014 works well to report concentration of 3-oxo-hexanoyl-HSL inside E. coli cells. However, we do advice future teams to measure the response to various known concentrations of 3-oxo-hexanoyl-HSL instead of with the luxI gene, to identify what concentration causes effective induction of the lux pR promoter. We highly recommend the use of BBa_K1913014 in conjunction with BBa_K546000, as we have found these parts work well together. In an attempt to streamline the use of these parts for future team, we created BBa_K1913005 (https://parts.igem.org/Part:BBa_K1913005), a composite part that includes both BBa_K546000 and BBa_K1913014.

Sequence and Features