Part:BBa_K4294801
OpenLoop
This is a circuit of the LuxR quorum sensing regulator in its simplest form.
Usage and Biology
This induction system is a quorum sensing system derived Vibrio fisheri [1] It is generally used in synthetic biology to programming communication between bacterial populations and is well characterized in literature.
Circuit Design
LuxR is constitutively expressed. When the concentration of OC6 increases, it binds with LuxR, LuxR forms stable dimmers and binds to its binding site (lux box), upstream of the -35 region of the Plux promoter, activating the transcription of the downstream output. tr
Figure 1: Flowchart of the Open Loop circuit
Figure 1: OL genetic circuit overview. LuxR is constitutively expressed under the control of the constitutive BBa_J23105 promoter and BBa_B0034 RBS. Output (mNeonGreen) production is controlled by the Plux inducible promoter and BBa_B0034 RBS.
Figure 1: OL at is “OFF” state (‘“0”)
Figure 1: OL at its “ON” state (“1”)
Characterisation
We constructed this circuit in both a medium (ColE1 origin of replication) and a low (p15a origin of replication) copy number pTU2 plasmid vector so as to choose the copy number that provides the most suitable dynamic range, leakiness and steepness. The medium copy number plasmid was chosen and used for the rest of the constructs.
Figure 1: pTU2 (ColE1) vector with OL circuit.
Figure 1: pTU2 (p15a) vector with the OL circuit.
Measurment
In our design the downstream output was mNeonGreen fluorescent protein. To quantify this output we measured the fluorescence using microplate reader FlexStation3 (Molecular Devices) with excitation wavelength 476nm and emission wavelength 547nm as suggested from the manufacturer. We conducted measurements in different time points after the induction with OC6, using different concentrations of the inducer. (photo από induction with p15a +cole1)
Figure 1: Induction of BL21 OL pTU2-RFP colE1 or. with the following OC6 concentrations (μΜ): 100, 10, 1, 0.1, 0.01, 0.001, 0.0001. The uninduced cells are represented by the concentration value 0.0000001 for the diagram purposes.
Figure 1: Induction of BL21 OL pTU2-RFP p15A or. with the following OC6 concentrations (μΜ): 100, 10, 1, 0.1, 0.01, 0.001, 0.0001. The uninduced cells are represented by the concentration value 0.0000001 for the diagram purposes.
References
[1] Dunlap P. V. (1999). Quorum regulation of luminescence in Vibrio fischeri. Journal of molecular microbiology and biotechnology, 1(1), 5–12. [2] R.M. Davis,Front. Bioeng. Biotechnol., 10 March 2015 Sec. Synthetic Biology https://doi.org/10.3389/fbioe.2015.00030
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 985
Illegal NheI site found at 1008 - 21INCOMPATIBLE WITH RFC[21]Illegal suffix found in sequence at 928
Illegal BglII site found at 916 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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