Difference between revisions of "Part:BBa K1216008"

Revision as of 13:40, 30 October 2013

Variant of the wild-type pLuxR promoter with lower sensitivity

Figure 8: Sequence alignment of the library member luxR variants.

BBa_K1216008: part of a P_LuxR collection of promoters with differing AHL sensitivity. This significantly increases usefulness of the Lux signaling system, as differential respones to cell concentrations are enabled. Further they can even be used to detect geometrical settings of colonies.

Usage and Biology

Figure 1: Overview of the Lux signaling system. With no LuxR-AHL complex bound, P_LuxL enables transcription of luxR whereas P_LuxR is shut off except for slight expression of the downstream operon. If AHL concentration rises inside the cell, P_LuxL is shut down and P_LuxR induced, yielding high expression of LuxI and thus further increase in AHL concentration, constituting positive feedback.

The Lux signaling system is based on positive feedback. The P_LuxL promoter drives expression of the upstream luxR gene. The promoter P_LuxR driver expression of the downstream operon including the LuxI AHL synthetase. The promoter encompasses a palindromic operator sequence to which a dimer of LuxR-AHL complexes binds to induce transcription. In absence of this complex, P_LuxL is active and thus LuxR is abundant. P_LuxR however is inactive, showing slight leakage, yielding a fairly low expression level of LuxI and other downstream gene products.

An important feature of AHL, the "signaling molecule" of the system, is that it diffuses freely through cell walls and surrounding media. This enables communication between cells in vincinity. When a certain cell concentration is reached, the diffusing AHL yields sufficiently high levels of the LuxR-AHL complex within cells. The direct consequence of this is high expression of downstream genes, importantly inculding LuxI (and not so importantly shutting down P_LuxL). Now the positive feedback loop is being closed by elevated synthesis of AHL which further increases LuxI synthesis in cells.

In V. Fischeri other downstream genes include a luciferase which is part of a symbiosis with marine animals. Other possibilities are proteins that facilitate biofilm formation and related phenomena.

In (synthetic) biology, this system can be used as a sender/reporter system by separating LuxI and LuxR, where LuxI is not under control of P_LuxR, rendering the system independent of cell concentration. LuxR/P_LuxR can then be used to drive expression of a myriad of other genes such as various reporter genes.

Sequence and Features