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Team:BIOTEC Dresden/Characterized Parts/BBa I13263
From 2010.igem.org
Part 17: I13263
This part is supposed to serve as a potential detection and quantification system for the concentration of the signal molecule acylated homoserine lactone (AHL) in the growth medium. As shown in the parts overview LuxR is constitutively expressed. In the presence of AHL a LuxR-AHL complex is formed activating lux pR and thereby the expression of eyfp. In characterization experiments the amount of AHL required for induction was analyzed. This part is characterized best and was consequently always used for later experiments.
Results
The graph 1.1 shows the YFP development over time of different concentrations of AHL, ranging from 10pM to 2µM. It can be seen that the fluorescence signal does not significantly change over time for AHL concentrations below 100nM and that the amount of YFP produced is the same for all concentrations above 300nM.
In the Graph 1.2 the fluorescence as a function of the AHL concentration after two hours of incubation is visualized. It can be seen that the YFP signal after 2 hours of incubation increases with increasing AHL concentrations but reaches a plateau at about 700nM.
Discussion:
Graph 1.1 shows nicely that the LB medium (dark blue) has a fluorescence signal of about 2800 raw fluorescence units (RFU). But since the fluorescence signal stayed constant over the time measured, this fluorescence does not qualitatively influence the data and can thus be ignored. Additionally it can be seen that the concentrations below 100nM do not lead to an increase in fluorescence, leading to the conclusion that a minimum concentration of 100nM outside of the bacteria is required for the induction of the LuxpR and in turn for the detectable expression of YFP. Furthermore, the graph shows that after 2h of incubation with AHL the amount of YFP detected can directly by related to the initial concentration of AHL.
Graph 1.2 on the other hand shows a logarithmic behavior of the transcriptional induction of the LuxpR promoter by AHL, which reaches its maximum induction at an AHL concentration of about 750nM.
Materials and methods:
The characterization was performed using a 96-well plate and a fluorescence plate reader. Bacteria supplied with the part BBa_I13263 were suspended in medium of a certain concentration of AHL ranging from 0.01 to 2000nM. The fluorescence was measured every 5 minutes using an excitation wavelength of 485nm and an emission wavelength of 535nm. For every fluorescence value, also the optical density at 612nm was measured. As a negative control the same measurements were done on uninduced bacteria and LB-medium without cells.
Additional information
For detailed information of the experimental set-up, please have a look at the protocols on our team-wiki. (Link) In case you want to know more about the normalization and negative controls, please have a look at this page: (Link)
