Difference between revisions of "Part:BBa K2771030"
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[[File:T--USP-Brazil--qs_cells.png|500px|thumb|none|alt=experiment 1.|Figure 1. Plate with only a droplet of high-concentration arabinose(top left) and plate slathered with the molecule (bottom right). Fluorescence shows that synthase can successfully produce 3OC6 HSL and it can, along with Lux R, activate a Lux promoter even at a distance]]
 
[[File:T--USP-Brazil--qs_cells.png|500px|thumb|none|alt=experiment 1.|Figure 1. Plate with only a droplet of high-concentration arabinose(top left) and plate slathered with the molecule (bottom right). Fluorescence shows that synthase can successfully produce 3OC6 HSL and it can, along with Lux R, activate a Lux promoter even at a distance]]
  
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We also tested the part in liquid culture assays, with 2.5mM arabinose, measuring the activity of a YFP reporter, to be normalized by a CFP constitutive expression. We showed that this part works, at least for promoters pLux and pLas:  
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We also tested the part in liquid culture assays, with 2.5mM arabinose, measuring the activity of a YFP reporter, to be normalized by a CFP constitutive expression. We showed that this part works, at least for the modified promoters pLux([https://parts.igem.org/Part:BBa_K2771000 BBa_K2771000]) and pLas([https://parts.igem.org/Part:BBa_K2771001 BBa_K2771001]):  
  
 
[[File:T--USP-Brazil--Lux_Las_assay.png|500px|thumb|none|alt=experiment 1.|Figure 2: Liquid culture assay with 2.5 mM induction at 0h. Extra detail in the experience page]]
 
[[File:T--USP-Brazil--Lux_Las_assay.png|500px|thumb|none|alt=experiment 1.|Figure 2: Liquid culture assay with 2.5 mM induction at 0h. Extra detail in the experience page]]
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We tested this construct's activity not just in LB, but also in M9 medium, again with promoters pLux and pLas

Revision as of 23:10, 17 October 2018


Lux inducible Synthase and constitutive Receiver

This part encodes for two genes in a quorum sensing signaling pathway, intended in our project for measurement of genetic crosstalk with quorum sensing responsive promoters. One of the genes, transcribing for LuxI, an Homoserine Lactone (HSL) Synthase, is transcribed by a promoter inducible by L-arabinose, permitting for controlled expression of a 3OC6 HSL signal. The other gene is constitutively expressed by an Andersen family promoter. If coupled to a gene with a promoter responsive to quorum sensing signals, this construct can create a response dependant on cell density and L-arabinose concentration. If there is a spatial difference in arabinose concentration, the HSL signal will diffuse in the medium and present a weaker response when farther to the signal origin.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
    Illegal NheI site found at 2031
    Illegal NheI site found at 2054
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1873
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 3009
    Illegal SapI site found at 961


USP-Brazil's Characterization

This part has shown to work. As a simple first proof-of-concept for this construct, we co-transformed it with a pLux promoter with a fluorescent reporter(BBa_K2771021), along with slathering the agar plate with high concentration arabinose or just putting a drop of that in a point of the plate, to induce the production of LuxI synthase and test for the quorum sensing distance-signaling mechanism. Controls not shown in photo, did not show fluorescence on the UV lightbox, while tests did:

experiment 1.
Figure 1. Plate with only a droplet of high-concentration arabinose(top left) and plate slathered with the molecule (bottom right). Fluorescence shows that synthase can successfully produce 3OC6 HSL and it can, along with Lux R, activate a Lux promoter even at a distance

We also tested the part in liquid culture assays, with 2.5mM arabinose, measuring the activity of a YFP reporter, to be normalized by a CFP constitutive expression. We showed that this part works, at least for the modified promoters pLux(BBa_K2771000) and pLas(BBa_K2771001):

experiment 1.
Figure 2: Liquid culture assay with 2.5 mM induction at 0h. Extra detail in the experience page

We tested this construct's activity not just in LB, but also in M9 medium, again with promoters pLux and pLas