Difference between revisions of "Part:BBa K546005"

Revision as of 19:17, 21 September 2011

Lux pL controlled LuxR + lux pR autoinducing LuxI (lva tag) + lux pR controlled GFP(lva tag).

This BioBrick composite part has an, colony wide, initially increasing and finally constant Green Fluorescent Protein (GFP) with a LVA Tag output.

Usage and Biology

This system consists of a positive and a negative feedback loop. The positive feedback loop is initiated when the constitutively expressed luxR forms a complex with AHL- which is produced by the protein LuxI. The complex up-regulates the expression of luxI, GFP and AiiA, at first exponentially increasing LuxI, GFP and AiiA levels because the maturation time of LuxI is shorter than that of AiiA. Then the negative feedback loop gets the upper hand: Once maturated, the enzyme AiiA degrades AHL faster than luxI can produce it. Because GFP is under the control of the same promoter as luxI it is highly expressed until AHL is rapidly being degraded by AiiA. Once the AHL concentration is lowered enough, the expression of GFP is halted. At this point the net change in GFP will thus become negative due to the degradation. Since AiiA expression is initiated by the luxR-AHL complex, the production of AiiA also halts which causes the net change of AiiA to become negative and this eventually causes the concentration of AiiA to become low enough to allow a net production of AHL again. Since AHL is a quorum sensing molecule all cells are synchronised in their oscillatory behavior.

Furthermore, this system implements two hybrid promoters: One controlling the transcriptional rate of luxI and the other one controlling the transcriptional rate of AiiA. These two promoters are respectively repressible by IPTG and aTc. Therefore, it should be possible to tune the period and the amplitude of the synchronized oscillations.

Safety Aspects

This BioBrick part produces an autoinducer that might interact with [http://2011.igem.org/Team:Wageningen_UR/Safety/One#Risk_Identification_of_BioBrick_System_Inside_the_Cell_Chassis some pathogens].

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 3909
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1101
Illegal BsaI.rc site found at 1828
Illegal BsaI.rc site found at 2086
Illegal BsaI.rc site found at 3189

@@ Line 7: / Line 7: @@
 ===Usage and Biology===
-LuxR is slightly produced by the sub-[https://parts.igem.org/Part:BBa_K546003 BioBrick part] K546003 at the beginning. Meanwhile also the mildly transcribed BioBrick part C0061 creates an increase in the [http://en.wikipedia.org/wiki/Autoinducer autoinducer] 3OC6HSL. As the LuxR and the autoinducer are formed simultaneously, a complex of these two forms -in all the cells of a colony. This complex initiates a positive and two times a negetive feedback loop. The gene luxI is part of the positive feedback loop and will be transcribed more often (resulting in a greater autoinducer production), but the promoter in front of the luxr gene will down regulate the formation of LuxR. The increase in and finally more or less equilibrium state of the LuxR-autoinducer complex is monitored by the Green Fluorescent Protein with a LVA Tag.
+This system consists of a positive and a negative feedback loop. The positive feedback loop is initiated when the constitutively expressed luxR forms a complex with AHL- which is produced by the protein LuxI. The complex  up-regulates the expression of luxI, GFP and AiiA, at first exponentially increasing LuxI, GFP and AiiA levels because the maturation time of LuxI is shorter than that of AiiA. Then the negative feedback loop gets the upper hand: Once maturated, the enzyme AiiA degrades AHL faster than luxI can produce it. Because GFP is under the control of the same promoter as luxI it is highly expressed until AHL is rapidly being degraded by AiiA. Once the AHL concentration is lowered enough, the expression of GFP is halted. At this point the net change in GFP will thus become negative due to the degradation. Since AiiA expression is initiated by the luxR-AHL complex, the production of AiiA also halts which causes the net change of AiiA to become negative and this eventually causes the concentration of AiiA to become low enough to allow a net production of AHL again. Since AHL is a quorum sensing molecule all cells are synchronised in their oscillatory behavior.
+Furthermore, this system implements two hybrid promoters: One controlling the transcriptional rate of luxI and the other one controlling the transcriptional rate of AiiA. These two promoters are respectively repressible by IPTG and aTc. Therefore, it should be possible to tune the period and the amplitude of the synchronized oscillations.
 ===Safety Aspects===