Difference between revisions of "Part:BBa K1216007"

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[[File:facsdatag1.jpg|700px|center|Thumb|<b>Figure 2. Dose response comparison between [OHHL] induction in liquid culture and on agar plates, of the J09855 construct using the BBa_K2126007 promoter and a GFP reporter analyzed at different [OHHL] in the FACS single cell analysis. The black curve shows the response of to OHHL in liquid culture, the orange curve shows the response to OHHL on agar plates]]
 
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Revision as of 18:42, 3 October 2013

Variant of the wild-type pLuxR promoter with lower sensitivity

BBa_K1216007 is a variant of the wild-type pLuxR promoter with a lowered sensitivity for LuxR-AHL.

Comparison of BBa_R0062 to the mutant

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Site directed mutagenesis of the BBa_R0062 pLuxR wild type promoter to obtain the pLuxR variant BBa_K2126007

Figure 1. Site of the directed mutagenesis in the luxbox.As indicated you can see the mutations of the pLuxR variant compared to the wild type in red. The positions which were mutated randomly are indicated in red-underlined


Our goal was to achieve mutations in the luxbox of the pLuxR BBa_R0062 promoter to shift the dose response curve of the promoter depending on the OHHL (3-oxo-hexanoyl-l-homoserine-l-lactone) concentration.
We did site directed mutagenisis of specific sites of the luxbox according to the results of the literature ()
We mutated the promoter directly in the J09855 construct and added a GFP reporter to be able to screen for differences in the dose response curves. In the end we isolate one mutated promoter which shows a 300'000 fold shift in sensivity. For more details see : Characterization of the pLuxR variant dose response to OHHL using plate reader analysis and FACS single cell analysis

Characterization of the pLuxR variant dose response to OHHL using plate reader analysis and FACS single cell analysis

File:Platereader.jpg

Figure 2. Dose response comparison between [OHHL] induction in liquid culture and on agar plates, of the J09855 construct using the BBa_K2126007 promoter and a GFP reporter analyzed at different [OHHL] in the FACS single cell analysis. The black curve shows the response of to OHHL in liquid culture, the orange curve shows the response to OHHL on agar plates

AgaLiG1.jpg