Difference between revisions of "Part:BBa K1913014"

(Usage and Biology)
 
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https://static.igem.org/mediawiki/2016/a/a1/T--Wageningen_UR--2plasmid_quorum_sensing_graph.jpg
 
https://static.igem.org/mediawiki/2016/a/a1/T--Wageningen_UR--2plasmid_quorum_sensing_graph.jpg
<p> Figure 1. Fluorescence (excitation: 480nm, emission 510nm, bandwidth: 9nm) and absorbance data for <i>E. coli</i> quorum sensing strains. Error bars indicate standard error of the mean. Continuous line: Fluorescence/OD<sub>600</sub>; Dashed line: Absorbance at 600nm (OD<sub>600</sub>). 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/OD<sub>600</sub> data were normalized to the highest Fluorescence/OD<sub>600</sub> 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. </p><br><br>
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<p><b> Figure 1. Fluorescence (excitation: 480nm, emission 510nm, bandwidth: 9nm) and absorbance data for <i>E. coli</i> quorum sensing strains. Error bars indicate standard error of the mean. Continuous line: Fluorescence/OD<sub>600</sub>; Dashed line: Absorbance at 600nm (OD<sub>600</sub>). 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/OD<sub>600</sub> data were normalized to the highest Fluorescence/OD<sub>600</sub> 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.</b> </p><br><br>
  
 
https://static.igem.org/mediawiki/2016/9/9c/T--Wageningen_UR--complete_QS_2plasmid_graph.jpg
 
https://static.igem.org/mediawiki/2016/9/9c/T--Wageningen_UR--complete_QS_2plasmid_graph.jpg
<p> Figure 2. Fluorescence and absorbance data for <i>E. coli</i> quorum sensing strains. Error bars indicate standard error of the mean. Continuous line: Fluorescence/OD600; Dashed line: Absorbance at 600nm (OD<sub>600</sub>). 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/OD<sub>600</sub> data were normalized to the highest Fluorescence/OD<sub>600</sub> 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 OD<sub>600</sub> and  Fluorescence/OD<sub>600</sub> values. </p> <br><br>
+
<p> <b>Figure 2. Fluorescence and absorbance data for <i>E. coli</i> quorum sensing strains. Error bars indicate standard error of the mean. Continuous line: Fluorescence/OD600; Dashed line: Absorbance at 600nm (OD<sub>600</sub>). 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/OD<sub>600</sub> data were normalized to the highest Fluorescence/OD<sub>600</sub> 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 OD<sub>600</sub> and  Fluorescence/OD<sub>600</sub> values. </b></p> <br><br>
  
 
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 OD<sub>600</sub>) 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.  
 
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 OD<sub>600</sub>) 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.  

Latest revision as of 21:31, 19 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).



T--Wageningen_UR--2plasmid_quorum_sensing_graph.jpg

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.



T--Wageningen_UR--complete_QS_2plasmid_graph.jpg

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 720